Primers and methods for the detection and discrimination of nucleic acids

ABSTRACT

The present invention provides novel primers and methods for the detection of specific nucleic acid sequences. The primers and methods of the invention are useful in a wide variety of molecular biology applications and are particularly useful in allele specific PCR.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is related to U.S. Provisional PatentApplication Nos. 60/139,890, filed Jun. 22, 1999, and 60/175,959, filedJan. 13, 2000, both of which are specifically incorporated herein byreference in their entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the field of molecular biology. Inparticular, the present invention relates to novel primers for use inthe detection and discrimination of nucleic acids. The novel primers ofthe present invention will find broad applicability in the field ofmolecular biology and, in particular, in the detection of products innucleic acid amplification reactions and in the discrimination betweenalleles of a given target gene.

2. Related Art

Assays capable of detecting and quantifying the presence of a particularnucleic acid molecule in a sample are of substantial importance inforensics, medicine, epidemiology and public health, and in theprediction and diagnosis of disease. Such assays can be used, forexample, to identify the causal agent of an infectious disease, topredict the likelihood that an individual will suffer from a geneticdisease, to determine the purity of drinking water or milk, or toidentify tissue samples. The desire to increase the utility andapplicability of such assays is often frustrated by assay sensitivity.Hence, it would be highly desirable to develop more sensitive detectionassays.

Nucleic acid detection assays can be predicated on any characteristic ofthe nucleic acid molecule, such as its size, sequence and, if DNA,susceptibility to digestion by restriction endonucleases. Thesensitivity of such assays may be increased by altering the manner inwhich detection is reported or signaled to the observer. Thus, forexample, assay sensitivity can be increased through the use ofdetectably labeled reagents. A wide variety of such labels have beenused for this purpose. Detectable labels include, for example,radioactive isotopes, fluorescent labels, chemiluminescent labels,bioluminescent labels and enzyme labels. U.S. Pat. No. 4,581,333describes the use of enzyme labels to increase sensitivity in adetection assay. Radioisotopic labels are disclosed in U.S. Pat. Nos.4,358,535, and 4,446,237. Fluorescent labels (EP 144,914), chemicallabels (U.S. Pat. Nos. 4,582,789 and 4,563,417) and modified bases (EP119,448) have also been used in an effort to improve the efficiency withwhich detection can be observed.

Although the use of highly detectable labeled reagents can improve thesensitivity of nucleic acid detection assays, the sensitivity of suchassays remains limited by practical problems which are largely relatedto non-specific reactions which increase the background signal producedin the absence of the nucleic acid the assay is designed to detect. Inresponse to these problems, a variety of detection and quantificationmethods using DNA amplification have been developed.

Many current methods of identification and quantification of nucleicacids rely on amplification and/or hybridization techniques. While manyof these involve a separation step, several that allow detection ofnucleic acids without separating the labeled primer or probe from thereaction have been developed. These methods have numerous advantagescompared to gel-based methods, such as gel electrophoresis, and dot-blotanalysis, for example, and require less time, permit high throughput,prevent carryover contamination and permit quantification through realtime detection. Most of these current methods are solution-basedfluorescence methods that utilize two chromophores. These methodsutilize the phenomena of fluorescence resonance energy transfer (FRET)in which the energy from an excited fluorescent moiety is transferred toan acceptor molecule when the two molecules are in close proximity toeach other. This transfer prevents the excited fluorescent moiety fromreleasing the energy in the form of a photon of light thus quenching thefluorescence of the fluorescent moiety. When the acceptor molecule isnot sufficiently close, the transfer does not occur and the excitedfluorescent moiety may then fluoresce. The major disadvantages ofsystems based on FRET are the cost of requiring the presence of twomodified nucleotides in a detection oligonucleotide and the possibilitythat the efficiency of the quenching may not be sufficient to provide ausable difference in signal under a given set of assay conditions. Otherknown methods which permit detection without separation are:luminescence resonance energy transfer (LRET) where energy transferoccurs between sensitized lanthanide metals and acceptor dyes (Selvin,P. R., and Hearst, J. D., Proc. Natl. Acad. Sci. USA 91:10024-10028(1994)); and color change from excimer-forming dyes where two adjacentpyrenes can form an excimer (fluorescent dimer) in the presence of thecomplementary target, resulting in a detectably shifted fluorescencepeak (Paris, P. L. et al., Nucleic Acids Research 26:3789-3793 (1998)).

Various methods are known to those skilled in the art for theamplification of nucleic acid molecules. In general, a nucleic acidtarget molecule is used as a template for extension of anoligonucleotide primer in a reaction catalyzed by polymerase. Forexample, Panet and Khorana (J. Biol. Chem. 249:5213-5221 (1974))demonstrate the replication of deoxyribopolynucleotide templates boundto cellulose. Kleppe et al., (J. Mol. Biol. 56:341-361 (1971)) disclosethe use of double and single-stranded DNA molecules as templates for thesynthesis of complementary DNA.

Other known nucleic acid amplification procedures include transcriptionbased amplification systems (Kwoh, D. et al., Proc. Natl. Acad. Sci. USA86:1173 (1989); PCT appl. WO 88/10315). Schemes based on ligation(“Ligation Chain Reaction”, “LCR”) of two (or more) oligonucleotides inthe presence of a target nucleic acid having a sequence complementary tothe sequence of the product of the ligation reaction have also been used(Wu, D. Y. et al., Genomics 4:560 (1989)). Other suitable methods foramplifying nucleic acid based on ligation of two oligonucleotides afterannealing to complementary nucleic acids are known in the art.

PCT appl. WO 89/06700 discloses a nucleic acid sequence amplificationscheme based on the hybridization of a promoter/primer sequence to atarget single-stranded DNA (“ssDNA”) followed by transcription of manyRNA copies of the sequence. This scheme is not cyclic; i.e. newtemplates were not produced from the resultant RNA transcripts.

EP 0 329,822 discloses an alternative amplification procedure termedNucleic Acid Sequence-Based Amplification (NASBA). NASBA is a nucleicacid amplification process comprising cyclically synthesizingsingle-stranded RNA (“ssRNA”), ssDNA, and double-stranded DNA (dsDNA).The ssRNA is a first template for a first primer oligonucleotide, whichis elongated by reverse transcriptase (RNA dependent DNA polymerase).The RNA is then removed from resulting DNA:RNA duplex by the action ofribonuclease H (RNase H, an RNase specific for RNA in a duplex witheither DNA or RNA). The resultant ssDNA is a second template for asecond primer. The second primer includes the sequences of an RNApolymerase promoter (exemplified by T7 RNA polymerase) located 5′ to theprimer sequence which hybridizes to the ssDNA template. This primer isthen extended by a DNA polymerase (exemplified by the large “Klenow”fragment of E. coli DNA polymerase I), resulting in the production of adouble-stranded DNA (“dsDNA”) molecule, having a sequence identical tothat of the portion of the original RNA located between the primers andhaving additionally, at one end, a promoter sequence. This promotersequence can be used by the appropriate RNA polymerase to make many RNAcopies of the DNA. These copies can then re-enter the cycle leading tovery swift amplification. With proper choice of enzymes, thisamplification can be done isothermally without addition of enzymes ateach cycle. Because of the cyclical nature of this process, the startingsequence can be chosen to be in the form of either DNA or RNA.

U.S. Pat. No. 5,455,166 and EP 0 684 315 disclose a method called StrandDisplacement Amplification (SDA). This method is performed at a singletemperature and uses a combination of a polymerase, an endonuclease anda modified nucleoside triphosphate to amplify single-stranded fragmentsof the target DNA sequence. A target sequence is fragmented, madesingle-stranded and hybridized to a primer that contains a recognitionsite for an endonuclease. The primer:target complex is then extendedwith a polymerase enzyme using a mixture of nucleoside triphosphates,one of which is modified. The result is a duplex molecule containing theoriginal target sequence and an endonuclease recognition sequence. Oneof the strands making up the recognition sequence is derived from theprimer and the other is a result of the extension reaction. Since theextension reaction was performed using a modified nucleotide, one strandof the recognition site is modified and resistant to endonucleasedigestion. The resultant duplex molecule is then contacted with anendonuclease which cleaves the unmodified strand causing a nick. Thenicked strand is extended by a polymerase enzyme lacking 5′-3′exonuclease activity resulting in the displacement of the nicked strandand the production of a new duplex molecule. The new duplex molecule canthen go through multiple rounds of nicking and extension producingmultiple copies of the target sequence.

The most widely used method of nucleic acid amplification is thepolymerase chain reaction (PCR). A detailed description of PCR isprovided in the following references: Mullis, K. et al., Cold SpringHarbor Symp. Quant. Biol. 51:263-273 (1986); European Patent (EP)50,424; EP 84,796; EP 258,017; EP 237,362; EP 201,184; U.S. Pat. No.4,683,202; U.S. Pat. No. 4,582,788; and U.S. Pat. No. 4,683,194. In itssimplest form, PCR involves the amplification of a targetdouble-stranded nucleic acid sequence. The double-stranded sequence isdenatured and an oligonucleotide primer is annealed to each of theresultant single strands. The sequences of the primers are selected sothat they will hybridize in positions flanking the portion of thedouble-stranded nucleic acid sequence to be amplified. Theoligonucleotides are extended in a reaction with a polymerase enzyme,nucleotide triphosphates and the appropriate cofactors resulting in theformation of two double-stranded molecules each containing the targetsequence. Each subsequent round of denaturation, annealing and extensionreactions results in a doubling of the number of copies of the targetsequence as extension products from earlier rounds serve as templatesfor subsequent replication steps. Thus, PCR provides a method forselectively increasing the concentration of a nucleic acid moleculehaving a particular sequence even when that molecule has not beenpreviously purified and is present only in a single copy in a particularsample. The method can be used to amplify either single ordouble-stranded nucleic acids. The essence of the method involves theuse of two oligonucleotidcs to serve as primers for the templatedependent, polymerase mediated replication of the desired nucleic acidmolecule.

PCR has found numerous applications in the fields of research anddiagnostics. One area in which PCR has proven useful is the detection ofsingle nucleotide mutations by allele specific PCR (ASPCR) (see forexample, U.S. Pat. No. 5,639,611 inventors Wallace, et al. and U.S. Pat.No. 5,595,890 inventors Newton, et al.). As originally described by Wu,et al. (Proceedings of the National Academy of Sciences, USA,86:2757-2760, 1989), ASPCR involves the detection of a single nucleotidevariation at a specific location in a nucleic acid molecule by comparingthe amplification of the target using a primer sequence whose3′-terminal nucleotide is complementary to a suspected variantnucleotide to the amplification of the target using a primer in whichthe 3′-terminal nucleotide is complementary to the normal nucleotide. Inthe case where the variant nucleotide is present in the target,amplification occurs more efficiently with the primer containing the3′-nucleotide complementary to the variant nucleotide while in the casewhere the normal nucleotide is present in the target, amplification ismore efficient with the primer containing 3′-nucleotide complementary tothe normal nucleotide.

While this technology can be used to identify single nucleotidesubstitutions in a nucleic acid, it nonetheless suffers from somedrawbacks in practical applications. The difference in efficiency ofamplification between the primers may not be sufficiently large topermit easily distinguishing between the normal nucleotide and themutant nucleotide. When the mismatched primer is extended with asignificant frequency in the earlier rounds of the amplification, theremay not be a large difference in the amount of product present in thelater rounds. This problem requires careful selection of the number ofamplification cycles and reaction conditions. An additional problem withthis methodology is presented by the detection step after theamplification. In general, this is accomplished by separating thereaction products by electrophoresis and then visualizing the products.The imposition of a separation step dramatically increases the time andexpense required for conducting this type of analysis. In order toobviate the need for a separation step, various FRET based solutionphase methods of detection have been used. These methods suffer from thedrawbacks discussed above.

Whether detection of a given nucleic acid target sequence is to be donewith or without amplification of the nucleic acid sample containing thetarget sequence, there remains a need in the art for more sensitive andmore discriminating methods of detecting a target nucleic acid sequence.

Methods for detecting nucleic acid amplification products commonly usegel electrophoresis, which separates the amplification product from theprimers on the basis of a size differential. Alternatively amplificationproducts can be detected by immobilization of the product, which allowsone to wash away free primer (for example, in dot-blot analysis) andhybridization of specific probes by traditional solid phasehybridization methods. However, several methods for monitoring theamplification process without prior separation of primer or probes havebeen described. All of these methods are based on FRET.

One method, described in U.S. Pat. No. 5,348,853 and Wang et al., Anal.Chem. 67:1197-1203 (1995), uses an energy transfer system in whichenergy transfer occurs between two fluorophores on the probe. In thismethod, detection of the amplified molecule takes place in theamplification reaction vessel, without the need for a separation step.The Wang et al. method uses an “energy-sink” oligonucleotidecomplementary to the reverse primer. The “energy-sink” andreverse-primer oligonucleotides have donor and acceptor labels,respectively. Prior to amplification, the labeled oligonucleotides forma primer duplex in which energy transfer occurs freely. Then, asymmetricPCR is carried out to its late-log phase before one of the targetstrands is significantly overproduced.

A second method for detection of amplification product without priorseparation of primer and product is the 5′ nuclease PCR assay (alsoreferred to as the TAQMAN™ assay) (Holland et al., Proc. Natl. Acad.Sci. USA 88:7276-7280 (1991); Lee et al., Nucleic Acids Res.21:3761-3766 (1993)). This assay detects the accumulation of a specificPCR product by hybridization and cleavage of a doubly labeledfluorogenic probe (the “TAQMAN” probe) during the amplificationreaction. The fluorogenic probe consists of an oligonucleotide labeledwith both a fluorescent reporter dye and a quencher dye. During PCR,this probe is cleaved by the 5′-exonuclease activity of DNA polymeraseif it hybridizes to the segment being amplified. Cleavage of the probegenerates an increase in the fluorescence intensity of the reporter dye.In the TAQMAN assay, the donor and quencher are preferably located onthe 3′ and 5′-ends of the probe, because the requirement that 5′-3hydrolysis be performed between the fluorophore and quencher may be metonly when these two moieties are not too close to each other (Lyamichevet al., Science 260:778-783 (1993)).

Another method of detecting amplification products (namely MOLECULARBEACONS) relies on the use of energy transfer using a “beacon probe”described by Tyagi and Kramer (Nature Biotech. 14:303-309 (1996)). Thismethod employs oligonucleotide hybridization probes that can formhairpin structures. On one end of the hybridization probe (either the 5′or 3′ end) there is a donor fluorophore, and on the other end, anacceptor moiety. In the case of the Tyagi and Kramer method, thisacceptor moiety is a quencher, that is, the acceptor absorbs energyreleased by the donor, but then does not itself fluoresce. Thus when thebeacon is in the open conformation, the fluorescence of the donorfluorophore is detectable, whereas when the beacon is in hairpin(closed) conformation, the fluorescence of the donor fluorophore isquenched. When employed in PCR, the beacon probe, which hybridizes toone of the strands of the PCR product, is in “open conformation,” andfluorescence is detected, while those that remain unhybridized will notfluoresce. As a result, the amount of fluorescence will increase as theamount of PCR product increases, and thus may be used as a measure ofthe progress of the PCR.

Another method of detecting amplification products, which relies on theuse of energy transfer is the SUNRISE PRIMER method of Nazarenko et al.(Nucleic Acids Research 25:2516-2521 (1997); U.S. Pat. No. 5,866,336).SUNRISE PRIMERS are based on FRET and other mechanisms ofnon-fluorescent quenching. SUNRISE PRIMERS consist of a single strandedprimer with a hairpin structure at its 5′ end. The hairpin stem islabeled with a donor/quencher pair. The signal is generated upon theunfolding and replication of the hairpin sequence by polymerase.

While there is a body of literature on use of fluorescent labelednucleic acids in a variety of applications involving nucleic acidhybridization or nucleic acid amplification, the majority ofapplications involve separation of unhybridized probes or unincorporatedprimers, followed by detection. None of these methodologies, describe ordiscuss real time detection of probes or primers, or changes in thefluorescence properties of a fluorescently labeled oligonucleotide uponhybridization or incorporation into amplified product. The surprisingand novel finding of the present invention is based, in part, on themeasurement of a change in one or more of the fluorescent properties oflabeled probes or primers upon becoming double-stranded.

The present invention thus solves the problem of detecting nucleicacids, in particular amplification and/or synthesis products, byproviding methods for detecting such products that are adaptable to manymethods for amplification or synthesis of nucleic acid sequences andthat greatly decrease the possibility of carryover contamination. Thecompounds and methods of the invention provide substantial improvementsover those of the prior art. First, they permit detection of theamplification or synthesis products without prior separation ofunincorporated fluorescent labeled oligonucleotides. Second, they allowdetection of the amplification or synthesis product directly, byincorporating the labeled oligonucleotide into the product. Third, theydo not require labeling of oligonucleotides with two different compounds(like FRET-based methods), and thus, simplify the production of thelabeled oligonucleotides.

SUMMARY OF THE INVENTION

The present invention provides oligonucleotides that may comprise one ormore modifications internally, and/or, at or near the 3′ and/or 5′termini. Suitable modifications include, but are not limited to, theinclusion of labels, the inclusion of specificity enhancing groups, theinclusion of quenching moieties and the like. The oligonucleotides ofthe present invention may also comprise one or more sequencescomplementary to all or a portion of a target or template sequence ofinterest. In some embodiments, the oligonucleotides of the presentinvention may be in the form of a hairpin. Hairpin oligonucleotides maybe modified or un-modified. Hairpin oligonucleotides of the presentinvention may contain one or more single stranded regions at or near thestem of the hairpin and may be blunt ended or comprise overhangingsequences on the 3′ and/or 5′-ends. The hairpin oligonucleotides of thepresent invention may also contain any number of stem and loopstructures at any location in the oligonucleotide. In some preferredembodiments, the oligonucleotides of the present invention may be usedfor the detection and/or discrimination of target or template nucleicacid molecules by methods involving primer extension including, but notlimited to, nucleic acid synthesis and amplification (e.g. PCR) as wellas by other methods involving hybridization of a probe and/or primer.The oligonucleotides of the present invention may be used with anyextension reaction known to those skilled in the art. Such extensionreactions include, but are not limited to, extension of a primer on aDNA template using a DNA polymerase to produce a complementary DNAstrand and extension of a primer on an RNA template using a reversetranscriptase to produce a complementary DNA strand. Theoligonucleotides of the present invention may also be used indetection/discrimination of target or template nucleic acid moleculesusing methods involving hybridization of one or more of theoligonucleotides of the invention to one or more target nucleic acidmolecules of interest.

In one aspect, oligonucleotides of the invention may comprise one ormultiple labels (e.g. detectable labels), which may be the same ordifferent. In some preferred embodiments, the labels may be fluorescentmoieties. Labeled oligonucleotides of the invention may be used todetect the presence or absence of or to quantify the amount of nucleicacid molecules in a sample by, for example, hybridization of sucholigonucleotides to such nucleic acid molecules. Optionally, sucholigonucleotides may be extended in a synthesis and/or amplificationreaction and detection/quantification may be accomplished during orafter such reactions. In accordance with one aspect of the invention,such detection/quantification is based on the observation that thelabeled oligonucleotides in double-stranded form have a detectablechange in one or more properties (preferably a fluorescent property)compared to the oligonucleotides in single-stranded form. In anotheraspect of the invention, a change in a detectable property (preferably afluorescent property) upon extension of the oligonucleotide of theinvention is used to detect/quantify a target/template nucleic acid.Fluorescent properties in which a change may be detected include, butare not limited to, fluorescent intensity (increase or decrease),fluorescent polarization, fluorescence lifetime and quantum yield offluorescence. Thus, hybridization and/or extension of the labeledoligonucleotides of the invention to a nucleic acid molecule to bedetected/quantified results in a detectable change in one or more of thelabels used and, in particular, when using fluorescent labels, adetectable change in one or more fluorescent properties. In this aspectof the invention, multiple different oligonucleotides may be used todetect multiple different target sequences in the same sample (e.g.multiplexing) and such different oligonucleotides may be differentiallylabeled to allow simultaneous and/or sequential detection of themultiple target sequences.

In another aspect, the present invention provides modifiedoligonucleotides comprising one or more specificity enhancing groups. Insome preferred embodiments, oligonucleotides of the present inventionmay be provided with one or more specificity enhancing groups thatrender such oligonucleotides substantially less extendable, for examplein a synthesis or amplification reaction, when the 3′-most nucleotide ofthe oligonucleotide is not base paired with a target or template nucleicacid sequence. In some embodiments, the specificity enhancing group maybe placed at or near the 3′most nucleotide of the oligonucleotide. Thespecificity enhancing group may be attached to the oligonucleotide usingany methodology known to those of skill in the art and may be attachedto the oligonucleotide via a linker group. Such linker groups may be ofvarying length and chemical composition, i.e., hydrophobicity, chargeetc. The specificity enhancing groups of the present invention may beattached to any part of the nucleotide to be modified, i.e., base, sugaror phosphate group. Specificity enhancing groups of the presentinvention may be or include detectable groups, including but not limitedto, fluorescent groups, chemiluminescent, radiolabeled groups and thelike. In some embodiments, the specificity enhancing groups of thepresent invention may be fluorescent groups which undergo a detectablechange in one or more fluorescent properties upon extension of theoligonucleotide or may be any other detectable label allowing detectionof the nucleic acid of interest. Preferably, the label exhibits adetectable change when the oligonucleotide of the invention is extendedin a synthesis or amplification reaction.

Oligonucleotides of the present invention may be in the form of ahairpin. The hairpins of the present invention preferably comprise atleast one stem structure and at least one loop structure. The sequenceswhich form the stem structure by base pairing may be of any length andpreferably contain at least a portion of a sequence complementary to atarget or template sequence. For example, the sequence of anoligonucleotide may be selected so as to form a hairpin structure at atemperature below the temperatures used in a synthesis or amplificationreaction by first selecting a sequence at least partially complementaryto a portion of a nucleic acid target or template sequence and thenadding one or more nucleotides to the 5′-end of the oligonucleotide thatare complementary to the nucleotides at the 3′-end of theoligonucleotide. At a reduced temperature, the complementary nucleotidesat the 3′ and 5′ ends can base pair forming a stem structure. The numberof complementary nucleotides to be added may be selected by determiningthe desired melting temperature of the stem structure. The meltingtemperature preferably is high enough that the oligonucleotide is in thehairpin structure when the reaction mixture is being prepared therebypreventing the oligonucleotide from mis-annealing to the target ortemplate nucleic acid molecule but low enough such that all or portionof the oligonucleotides are capable of assuming a linear structure andannealing to the target or template at the appropriate point in thesynthesis or amplification reaction. The selection of an appropriatemelting temperature for the stem structure is routine for those ofordinary skill in the art.

The oligonucleotides of the present invention may incorporate more thanone of the characteristics described above or combinations thereof. Forexample, an oligonucleotide may comprise one or more labels and/or oneor more specificity enhancing groups and/or one or more hairpinstructures.

In another aspect, one or more of the oligonucleotides of the presentinvention may be covalently or non-covalently attached to a support byany means known to those skilled in the art. Such support boundoligonucleotides may be used to carry out the methods of the presentinvention. For example, the detection or quantification of nucleic acidmolecules may be accomplished on a support and/or the synthesis oramplification of nucleic acids may be accomplished on a support. Such asupport may be solid or semisolid and may be made of any material knownto those skilled in the art.

In one aspect, the present invention provides for reaction mixtures orcompositions for use in a process for the synthesis and/or amplificationof one or more nucleic acid molecules complementary to all or a portionof one or more nucleic acid target or template molecules of interest. Insome preferred embodiments, the reaction mixture may comprise at least afirst and preferably a first and a second oligonucleotide primer of theinvention which primers may be the same or different and may contain thesame or different labels and/or specificity enhancing groups. Such firstprimer preferably comprises at least one sequence which is at leastpartially complementary to said target or template nucleic acid andwhich primes synthesis of a first extension product that iscomplementary to all or a portion of said target or template nucleicacid. Such second oligonucleotide primer preferably comprise a sequencewhich is at least partially complementary to all or a portion of saidfirst extension product and primes the synthesis of a second extensionproduct which is at least partially complementary to all or a portion ofsaid first extension product. In some embodiments, the reaction mixturemay comprise one or more oligonucleotide primers of the invention, whichmay be the same or different, and which may contain one or more of thesame or different labels and/or specificity enhancing groups. Forexample, the reaction mixture or composition may comprise more than oneoligonucleotide primer, wherein at least one of said primers is in theform of a hairpin and another is not. In another aspect, one primer maybe provided with a label that undergoes a detectable change in one ormore properties upon hybridization and/or extension while a secondprimer may be in the form of a hairpin and/or comprise a specificityenhancing group. In another aspect, both the first and the second primermay be in the form of a hairpin and may also comprise labels and/orspecificity enhancing groups as described above. Such reaction mixturesor compositions of the present invention may further comprise one ormore components selected from a group consisting of one or morenucleotides, one or more DNA polymerases, one or more reversetranscriptases, one or more buffers or buffering salts, one or moretarget or template molecules and one or more products produced by asynthesis/amplification reaction of the present invention. Thus, theinvention relates generally to compositions/reaction mixtures producedto carry out the invention and/or to composition/reaction mixturesresulting from carrying out the invention.

The present invention relates to a method for detecting the presence orabsence of a nucleic acid molecule or for quantifying the amount of anucleic acid molecule in a sample comprising:

-   (a) contacting a sample thought to contain one or more nucleic acid    molecules with one or more oligonucleotides of the invention; and-   (b) detecting the presence or absence or quantifying the amount of    nucleic acid molecules in said sample.    In some embodiments, the oligonucleotide may be labeled and the    detecting step may involve the detection of a change in one or more    fluorescent or other detectable properties of a the labeled    oligonucleotide of the present invention. In some embodiments, the    fluorescent property which undergoes a change is the intensity of    fluorescence. In some embodiments, an increase in fluorescence    intensity is detected.

Preferably the oligonucleotides of the invention are incubated underconditions sufficient to allow hybridization of such oligonucleotides tothe nucleic acid molecules in the sample. In a preferred aspect, thedetection or quantification step includes a comparison of a controlsample (without nucleic acid molecules present) to the sample containingnucleic acid molecules. Additional control samples containing knownamounts of nucleic acid molecules may be used in accordance with theinvention as a positive control for comparison purposes to determine theexact or approximate amount of the nucleic acid molecules present in theunknown sample.

In a related aspect, the invention relates to detection orquantification of nucleic acid molecules in a sample during or afternucleic acid synthesis or amplification. Thus, the invention relates toa method for detection or quantification of one or more nucleic acidmolecules in a sample comprising:

-   (a) mixing one or more nucleic acid templates or target nucleic acid    molecules of the sample with one or more oligonucleotides for the    invention;-   (b) incubating said mixture under conditions sufficient to    synthesize or amplify one or more nucleic acid molecules    complementary to all or a portion of said templates or target    molecules, wherein said synthesized or amplified nucleic acid    molecules comprise said oligonucleotide; and-   (c) detecting or quantifying said synthesized or amplified nucleic    acid molecules.    In some embodiments, the oligonucleotide may be labeled and the    detecting step may involve the detection of a change in one or more    fluorescent or other detectable properties of the labeled    oligonucleotide of the present invention. In some embodiments, the    fluorescent property which undergoes a change is the intensity of    fluorescence. In some embodiments, an increase in fluorescence    intensity is detected.

Conditions sufficient to synthesize or amplify one or more nucleic acidmolecules complementary to all or a portion of said templates or targetmolecules preferably comprise incubating the template/oligonucleotidemixture in the presence of one or more nucleotides and one or morepolymerases and/or reverse transcriptases (preferably DNA polymerasesand most preferably thermostable DNA polymerases). In a most preferredaspect, the amplification process used is polymerase chain reaction(PCR) or RT PCR, although other amplification methods may be used inaccordance with the invention. In this aspect of the invention, thedetection/quantification step may be accomplished during amplificationor synthesis or after synthesis or amplification is complete. Fordetection during an amplification reaction, a thermocycler capable ofreal time fluorescence detection may be used. Further, the nucleic acidsynthesis or amplification method preferably produces double-strandednucleic acid molecules (preferably double-stranded DNA/DNA or DNA/RNAmolecules) and the presence or absence or amount of such double-strandedmolecules may be determined by this method of the invention. In apreferred aspect, using the labeled oligonucleotides of the invention asa primer during synthesis or amplification, the labeled oligonucleotideprimer is incorporated into the synthesized or amplified moleculethereby creating a labeled product molecule (which may besingle-stranded or double-stranded). In another aspect, the synthesizedor amplified nucleic acid molecules produced in accordance with theinvention may contain one or more labels, which may be the same ordifferent. In a preferred aspect, the detection or quantification stepincludes a comparison of a control sample to the sample containing thetarget/template nucleic acid molecules of interest. Additional controlsamples containing known amounts of target/template may be used as apositive control for comparison purposes and/or to determine the exactor approximate amount of target/template in an unknown sample.

More specifically, the invention is directed to a method for amplifyinga double-stranded nucleic acid target molecule (e.g., DNA/DNA; RNA/RNA;or RNA/DNA), comprising:

-   (a) providing at least a first and a second primer, wherein said    first primer is complementary to a sequence within or at or near the    3′-termini of a first strand of said nucleic molecule and said    second primer is complementary to a sequence within or at or near    the 3′-termini of the second strand of said nucleic acid molecule;-   (b) hybridizing said first primer to said first strand and said    second primer to said second strand in the presence of one or more    of polymerases or reverse transcriptases, under conditions such that    a third nucleic acid molecule complementary to all or a portion of    said first strand and a fourth nucleic acid molecule complementary    to all or a portion of said second strand are synthesized;-   (c) denaturing said first and third strand, and said second and    fourth strands; and-   (d) repeating steps (a) to (c) one or more times, wherein one or    more of said primers are oligonucleotides of the present invention.    In some embodiments, at least one of the primers comprises a label    that undergoes a detectable change in one or more fluorescent or    other detectable properties upon hybridization and/or extension. In    some embodiments, at least one of the primers comprises a    specificity enhancing group that renders the primer substantially    less extendable when the 3′-nucleotide of the primer is not base    paired with the target molecule. In some embodiments, one or more of    the primers is in the form of a hairpin. In some embodiments, at    least one of the primers is in the form of a hairpin and further    comprises a detectable label and/or a specificity enhancing group.

In a further aspect, the present invention provides a method for thedirect detection of amplification or synthesis products in which thedetection may be performed without opening the reaction tube. Thisembodiment, the “closed-tube” format, reduces greatly the possibility ofcarryover contamination with amplification or synthesis products. Theclosed-tube method also provides for high throughput analysis of samplesand may be automated. The closed-tube format significantly simplifiesthe detection process, eliminating the need for post-amplification orpost-synthesis analysis such as gel electrophoresis or dot-blotanalysis.

In another aspect, the invention relates to a method for hybridizing orbinding one or more of the oligonucleotides of the invention with one ormore nucleic acid molecules of interest comprising:

-   (a) mixing one or more of said oligonucleotides with one or more of    said nucleic acid molecules; and-   (b) incubating said mixture under conditions sufficient to hybridize    or bind one or more of said oligonucleotides with one or more of    said nucleic acid molecules.    In a preferred aspect, at least one or more of the oligonucleotides    used in this method are hairpins and more preferably, the one or    more oligonucleotides are hairpin molecules comprising one or more    specificity enhancing groups and/or one or more labels.

The invention also relates to methods of synthesis or amplification ofone or more nucleic acid molecules comprising:

-   (a) mixing one or more templates or target nucleic acid molecules    with one or more oligonucleotides of the invention; and-   (b) incubating said mixture under conditions sufficient to    synthesize or amplify one or more nucleic acid molecules    complementary to all or a portion of said templates or target    molecules.    In a preferred aspect, the oligonucleotides are hairpins and more    preferably are hairpin molecules comprising one or more specificity    enhancing groups and/or one or more labels. Conditions sufficient to    synthesize or amplify one or more nucleic acid molecules    complementary to all or a portion of said templates or target    molecules preferably comprise incubating the    templates/oligonucleotide mixture (e.g., the    template-oligonucleotide complex) in the presence of one or more    nucleotides and one or more polymerases and/or one or more reverse    transcriptases (preferably DNA polymerases and most preferably    thermostable DNA polymerases). In a most preferred aspect, the    amplification process used is polymerase chain reaction (PCR) or RT    PCR, although other amplification methods may be used in accordance    with the invention. Further, the nucleic acid synthesis or    amplification methods preferably produces double stranded nucleic    acid molecules (preferably double stranded DNA/DNA or DNA/RNA    molecules). Use of the oligonucleotides of the invention allows for    more efficient synthesis and/or amplification of nucleic acid    molecules.

More specifically, the invention is directed to a method for amplifyinga double stranded nucleic acid target molecules comprising:

-   (a) providing a first and second primer, wherein said first primer    is complementary to a sequence within or at or near the 3′ termini    of the first strand of said nucleic acid molecule and said second    primer is complementary to a sequence within or at or near the 3′    termini of the second strand of said nucleic acid molecule;-   (b) hybriding said first primer to said first strand and said second    primer to said second strand in the presence of one or more    polymerases or reverse transcriptases, under conditions such that a    third nucleic acid molecule complementary to all or a portion of    said first strand and a fourth nucleic acid molecule complementary    to all or a portion of said second strand are synthesized;-   (c) denaturing said first and third strands, and said second and    first strands; and-   (d) repeating steps (a) to (c) one or more times, wherein one or    more of said primers are oligonucleotides of the present invention.    In one embodiment, the oligonucleotides of the invention used are    hairpins, and preferably are hairpins comprising one or more    specificity enhancing groups and/or one or more labels.

The invention also provides the embodiments of the above methods whereinthe nucleic acid molecule to bedetected/quantified/amplified/synthesized is an RNA or a DNA molecule,and wherein such molecule is either single-stranded or double-stranded.

The invention also provides the embodiments of the above methods whereinone or a number of the primers or oligonucleotides of the presentinvention comprise at least one nucleotide derivative. Examples of suchderivatives include, but are not limited to, a deoxyinosine residue, athionucleotide, a peptide nucleic acid and the like.

The invention also provides the embodiment of the above methods whereinthe nucleic acid target or template molecule is polyadenylated at its 3′end (e.g., poly(A) RNA or mRNA), and/or at least one of the primers oroligonucleotides of the invention contains a poly(T) sequence, and/or atleast one of the other of the primers or oligonucleotides of theinvention contains at least one deoxyinosine residue. In a relatedaspect, the template or target nucleic acid is an mRNA molecule, atleast one primer/oligonucleotide is labeled and comprises a poly(T)sequence and at least one primer/oligonucleotide comprises at least onedeoxyinosine residue.

As will be further appreciated, the labeled oligonucleotide sequences ofthe invention may be employed in other amplification methods, such asthose involving the application of PCR to the amplification of cDNA-endsderived from mRNAs using a single gene specific primer. Thus, labeledoligonucleotides of the invention can be used in methods such as“RT-PCR,” “5′-RACE,” “anchor PCR” and “one-sided PCR,” which facilitatethe capture of sequence from 5′-ends of mRNA. The methods of theinvention are adaptable to many methods for amplification of nucleicacid sequences, including PCR, LCR, SDA and NASBA, and otheramplification systems known to those of ordinary skill in the art.

In another aspect of the invention, the invention is directed to amethod for determining the activity or amount of a polymerase in asample, comprising amplifying a nucleic acid molecule, comprising:

-   (a) providing a first and second primer, wherein said first primer    is complementary to a sequence within or at or near the 3′-termini    of the first strand of said nucleic acid molecule and said second    primer is complementary to a sequence within or at or near the    3′-termini of the second strand of said nucleic acid molecule;-   (b) hybridizing said first primer to said first strand and said    second primer to said second strand in the presence of said    polymerase, under conditions such that a third nucleic acid molecule    complementary to all or a portion of said first strand and a fourth    nucleic acid molecule complementary to all or a portion of said    second strand are synthesized;-   (c) denaturing said first and third strand, and said second and    fourth strands; and-   (d) repeating steps (a) to (c) one or more times; and-   (e) detecting the amplification product, wherein at least one of the    primers are oligonucleotides of the present invention, and wherein    the amount of the amplification product produced is indicative of    the activity or amount of the polymerase.

In some embodiments, the amount of the amplification product produced isdetermined by detecting a change in one or more fluorescent or otherdetectable properties of an incorporated detectable label.

Generally, the invention thus relates to a method for determining theactivity or the amount of polymerase or reverse transcriptase in asample comprising:

-   (a) mixing a sample thought to contain a polymerase or reverse    transcriptase with one or more nucleic acid templates and one or    more labeled oligonucleotides of the invention;-   (b) incubating said mixture under conditions sufficient to allow    synthesis or amplification of one or more nucleic acid molecules    complementary to all or a portion of said templates, wherein said    synthesized or amplified nucleic acid molecules comprise said    oligonucleotides; and-   (c) determining the activity or amount of said polymerase or reverse    transcriptase in said sample based on detection of one or more    detectable labels.

In another aspect, the invention relates to quenching backgroundfluorescence during detection of nucleic acid molecules or polymerasesin accordance with the methods of the invention. In this aspect of theinvention, one or more quenching agents which bind one or more labeledsingle-stranded nucleic acid molecules are used to quench thefluorescence produced by such single-stranded molecules. In a preferredaspect, the quenching agent is specific for single-stranded moleculesand will not substantially interact with double-stranded labeled nucleicacid molecules. Thus, fluorescently labeled oligonucleotides of theinvention will be quenched or substantially quenched in the presence ofsuch agents. Upon interaction with the target molecule or duringamplification or synthesis reactions, the double-stranded nucleic acidmolecule formed which comprise the fluorescently labeledoligonucleotides of the invention will not substantially interact withsuch agents and thus will not be quenched by such agents. This aspect ofthe invention thus allows for reduced background fluorescence andenhanced detection of target nucleic acid molecules in the methods ofthe invention. Preferred quenchers for use in the invention include oneor more single-stranded binding proteins. In another aspect, suchquenching agents may include blocking oligonucleotides which contain oneor more quenchers, for example, DABCYL. In another aspect, the quenchingmoiety may be part of the oligonucleotide of the invention. For example,one or more quenching moieties may be incorporated into one or more stemstructures of the hairpin of the invention. Such stem structures mayalso incorporate one or more labels and in the hairpin configuration,the quenching moieties reduce the level of background activity of thelabel. Upon denaturation (unfolding) of the stem structure, thequenching of the label is reduced or prevented.

In another embodiment, the invention relates to a composition comprisingone or more labeled oligonucleotide of the invention, wherein the labelis a detectable label, and wherein the oligonucleotide is selected fromthe group consisting of DNA and RNA. The labeled oligonucleotides of theinvention may be primers and/or probes, depending on the use. Thecompositions of the invention may further comprise one or morecomponents selected from the group consisting of one or morepolymerases, one or more quenching agents, one or more nucleotides, oneor more nucleic acid molecules (which may be templates or nucleic acidmolecules which may comprise one or more oligonucleotides of theinvention), and one or more buffering salts.

In another embodiment of the invention, the label is a member of a FRETpair. In this embodiment, one or more labeled oligonucleotides of theinvention containing a single or multiple members of a FRET pairinternally, and/or, at or near the 3′ and/or 5′ end. In a preferredaspect, the labeled moiety is one or more fluorescent moieties whoseemission may then be measured to assess the progress of the reaction.

The present invention also relates to kits for the detection ormeasurement of nucleic acid synthesis or amplification products or forthe measurement or detection of nucleic acid molecules of the invention.Such kits may be diagnostic kits where the presence of the nucleic acidbeing amplified or synthesized is correlated with the presence orabsence of a disease or disorder. Kits of the invention may also be usedto detect or determine activity or amount of a polymerase in a sample.In addition, kits of the invention may be used to carry out synthesis,amplification or other extension reactions using the oligonucleotides ofthe invention. Preferred kits of the invention may comprise one or morecontainers (such as vials, tubes, and the like) configured to containthe reagents used in the methods of the invention and optionally maycontain instructions or protocols for using such reagents. The kits ofthe invention may comprise one or more components selected from thegroup consisting of one or more oligonucleotides of the invention(including probes and/or primers), one or more DNA polymerases, such asa thermostable polymerase, one or more reverse transcriptases, or anyother DNA or RNA polymerase, one or more agents capable of quenching oneor more of the labels, one or more buffers or buffering salts, one ormore nucleotides, one or more target/template molecules (which may usedfor determining reaction performance, i.e., control reactions) and otherreagents for analysis or further manipulation of the products orintermediates produced by the methods of the invention. Such additionalcomponents may include components used for cloning and/or sequencing andcomponents or equipment needed for the detection or quantification ofthe nucleic acid molecule of interest.

The invention also relates to any of the products or intermediates (e.g,nucleic acid molecules) produced by carrying out the methods of theinvention. The invention also relates to vectors or host cellscontaining such products or intermediates produced by the methods of theinvention. Introduction of such vectors into host cells may beaccomplished using any of the cloning and transformation techniquesknown to those skilled in the art.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic representation of the homogeneous/real-timedetection system of the invention. A change in one or more fluorescentor other detectable properties can be detected either through theincorporation of the labeled primer into the double-strandedamplification product (A), or through the direct hybridization of thelabeled probe to the nucleic acid target (B). In accordance with theinvention, the nucleic acid molecules detected or quantified can be asynthesized or amplified product or a nucleic acid molecule found innature. Such nucleic acid molecules may be single or double stranded andcan be RNA, DNA or RNA/DNA hybrids. In accordance with the invention,any one or more labels (which may be the same or different) may be used.

FIG. 2 is a graph of fluorescent intensity as a function of temperaturewhich shows the effect of hybridization on the fluorescence ofinternally (Panel A) and 5′-fluorescein labeled (Panel B)oligonucleotides. Labeled oligonucleotides were tested for fluorescenceunder different temperatures. Single-stranded (SS) or double-stranded(DS) oligonucleotides were melted as described in Example 4. For5′-labeled oligonucleotides, conversion from SS oligonucleotides to DSoligonucleotides caused a decrease in fluorescence, while for internallylabeled oligonucleotides, conversion from SS oligonucleotides to DScaused an increase in fluorescence.

FIG. 3 is a graph of fluorescent intensity as a function of wavelengthwhich shows fluorescence of 3′-TAMRA oligonucleotide in the presence ofcomplementary and non-complementary oligonucleotides. In presence ofcomplement (to create a double stranded molecule), the fluorescenceincreased compared to the single stranded form (see Example 5).

FIG. 4 is a graph of fluorescence as a function of wavelength whichshows the effect of hybridization on the fluorescence ofoligonucleotides 5′-labeled with fluorescein and BODIPY 530/550. In thepresence of the complement oligonucleotide (to create a double strandedmolecule), the fluorescence increased in case of BODIPY dye anddecreased in case of fluorescein.

FIG. 5 is a graph of fluorescent intensity as a function of the numberof cycles of amplification performed which shows quantitative PCR of IL4cDNA with an internally labeled primer (Panel A). PCR was performed asdescribed in Example 7. Data from ABI PRIZM™7700 Sequence Detector weretreated according to the manufacture's instructions with minormodifications. Panel 13 is a standard curve plotting the number ofcycles of amplification against the starting quantity of template DNA.

FIG. 6 is a graph of fluorescent intensity as a function of the numberof cycles of amplification performed which shows IL4 cDNA PCR with aprimer post-synthetically labeled with fluorescein. PCR was performed asdescribed in Example 8. Real-time amplification data were exported fromABI PRIZMT™ 7700 Sequence Detector in Excel.

FIG. 7 is a graph of fluorescent intensity as a function of the numberof cycles of amplification performed which shows detection of b-actincDNA by PCR with a primer internally labeled with fluorescein. PCR wasperformed as described in Example 9.

FIG. 8 is a graph of fluorescent intensity as a function of the numberof cycles of amplification performed which shows b-Actin cDNA PCR with aprimer internally labeled through a 5′-detection tail. PCR was performedas described in Example 10.

FIG. 9 is a schematic representation of allele specific PCR.

FIG. 10 is a photograph of an agarose gel showing the results of anallele specific PCR reaction comparing the primers of the presentinvention to standard primers.

FIG. 11 is a plot of fluorescence as a function of the number of cyclesof PCR performed in an allele specific PCR reaction comparing thehairpin primers of the present invention to standard linear primers.

FIG. 12 is a plot of fluorescence as a function of the number of cyclesof PCR performed in an allele specific PCR reaction comparing thehairpin primers of the present invention to standard linear primersusing a two step PCR reaction format.

FIG. 13 Panel A shows a bar graph of the fluorescence intensity obtainedat the end point of an allele specific PCR reaction using the primers ofthe present invention. Panel B is a photograph of the PCR tubes in whichthe allele specific reaction was conducted illuminated with ultravioletlight.

FIG. 14 is a photograph of an agarose gel showing the effects of targetDNA concentration on an allele specific PCR reaction using the primersof the present invention.

FIG. 15 is a photograph of an agarose gel showing the results of anallele specific reaction comparing the results obtained using Tsp DNApolymerase to Taq DNA polymerase using standard primers.

FIG. 16 is a photograph of an ethidium bromide stained agarose gelshowing the results of comparison of the hairpin oligonucleotides of thepresent invention to linear oligonucleotides in an amplificationreaction using varying amounts of template DNA. Panel A shows theamplification of a 3.6. kb fragment of the human beta-globin gene usinga first primer set. Panel B shows the amplification of a 3.6 kb fragmentof the human beta-globin gene using a second primer set.

FIG. 17 is a photograph of an ethidium bromide stained agarose gelshowing the results of comparison of the hairpin oligonucleotides of thepresent invention to linear oligonucleotides in an amplificationreaction to produce varying sized amplification products. Panel A showsthe amplification of a 1.3 kb fragment of the NF2 gene. Panel B showsthe amplification of a 1.6 kb fragment of the NF2 gene.

DETAILED DESCRIPTION OF THE INVENTION Definitions and Abbreviations

In the description that follows, a number of terms used in recombinant.DNA technology are extensively utilized. As used herein, the followingterms shall have the abbreviations indicated:

ASP, allele-specific polymerase chain reaction

bp, base pairs

DAB or DABCYL, 4-(4′-dimethylaminophenylazo) benzoic acid

EDANS, 5-(2′-aminoethyl) aminonapthalene-1-sulfonic acid

FAM or Flu, 5-carboxyfluorescein

JOE, 2′7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein

HPLC, high-performance liquid chromatography

NASBA, nucleic acid sequence-based amplification

Rhod, rhodamine

ROX, 6-carboxy-X-rhodamine

R6G, 6-carboxyrhodamine

TAMRA, N,N,N′,N′-tetramethyl-6-carboxyrhodamine

Amplification. As used herein, “amplification” refers to any in vitromethod for increasing the number of copies of a nucleotide sequence withthe use of a polymerase. Nucleic acid amplification results in theincorporation of nucleotides into a nucleic acid (e.g., DNA) molecule orprimer thereby forming a new nucleic acid molecule complementary to thenucleic acid template. The formed nucleic acid molecule and its templatecan be used as templates to synthesize additional nucleic acidmolecules. As used herein, one amplification reaction may consist ofmany rounds of nucleic acid synthesis. Amplification reactions include,for example, polymerase chain reactions (PCR). One PCR reaction mayconsist of 5 to 100 “cycles” of denaturation and synthesis of a nucleicacid molecule.

Specificity enhancing group. As used herein “specificity enhancinggroup” refers to any molecule or group of molecules that causes anoligonucleotide of the present invention to be substantially lessextendable when the 3′-most nucleotide of the oligonucleotide issubstantially not base paired with a nucleotide on the nucleic acidtarget/template molecule. Any type of group may be used. Preferredexamples include, but are not limited to, fluorescent groups, modifiednucleotides, small molecules, haptens and the like. Specificityenhancing groups may be attached at any position of the oligonucleotideso long as they make the oligonucleotide substantially less extendablewhen the 3′-terminal nucleotide of the oligonucleotide is substantiallynot base paired with the corresponding nucleotide of the target/templatenucleic acid. Such groups are preferably attached to the primer at ornear the 3′-end of the primer but may be attached at other positions aswell. Preferably, they are attached to one or more of the 25 basesadjacent to the 3′-end of the primer. In some preferred embodiments,such groups may be attached to one or more of the 20 bases adjacent tothe 3′-end of the oligonucleotide, or to the 15 bases adjacent to the3′-end or to the 10 base pairs adjacent to the 3′-end or, mostpreferably to one or more of the five bases adjacent to the 3′-end ofthe oligonucleotide. In addition, specificity enhancing groups may beattached to the 3′-most nucleotide so long as the presence of the groupdoes not prevent or inhibit the extension of the primer when the 3′-mostnucleotide of the primer is complementary to the correspondingnucleotide on the target/template molecule more than the extension isinhibited when the 3′-most nucleotide is substantially not base pairedto the target/template. Any group that can decrease the stability of theduplex formed by the primer and template when the 3′-most nucleotide ofthe primer is not complementary the corresponding nucleotide of thetarget/template and/or any group that can make a polymerase lessefficient at extending the 3′-end of the oligonucleotide when the3′-most nucleotide is not complementary to the corresponding nucleotideof the template/target may be used to practice the present invention. Insome embodiments, the specificity enhancing groups of the invention maybe modified nucleotides incorporated into the sequence of the primer.Such modifications may be made at the base, sugar or phosphate portionof the nucleotide and include but are not limited to phophothioatenucleotides, phosphonate nucleotides, peptide nucleic acids and thelike.

Polymerase. As used herein “polymerase” refers to any enzyme having anucleotide polymerizing activity. Polymerases (including DNA polymerasesand RNA polymerases) useful in accordance with the present inventioninclude, but are not limited to, Thermus thermophilus (Tth) DNApolymerase, Thermus aquaticus (Taq) DNA polymerase, Thermotoganeopolitana (Tne) DNA polymerase, Thermotoga maritima (Tma) DNApolymerase, Thermococcus litoralis (Tli or VENT™) DNA polymerase,Pyrococcus furiosus (Pfu) DNA polymerase, DEEPVENT™ DNA polymerase,Pyrococcus woosii (Pwo) DNA polymerase, Bacillus sterothermophilus (Bst)DNA polymerase, Bacillus caldophilus (Bca) DNA polymerase, Sulfolobusacidocaldarius (Sac) DNA polymerase, Thermoplasma acidophilum (Tac) DNApolymerase, Thermus flavus (Tfl/Tub) DNA polymerase, Thermus ruber (Tru)DNA polymerase, Thermus brockianus (DYNAZYME™) DNA polymerase,Methanobacterium thermoautotrophicum (Mth) DNA polymerase, mycobacteriumDNA polymerase (Mtb, Mlep), and mutants, and variants and derivativesthereof. RNA polymerases such as T3, T5 and SP6 and mutants, variantsand derivatives thereof may also be used in accordance with theinvention. Generally, any type I DNA polymerase may be used inaccordance with the invention although other DNA polymerases may be usedincluding, but not limited to, type III or family A, B, C etc. DNApolymerases.

Polymerases used in accordance with the invention may be any enzyme thatcan synthesize a nucleic acid molecule from a nucleic acid template,typically in the 5′ to 3′ direction. The nucleic acid polymerases usedin the present invention may be mesophilic or thermophilic, and arepreferably thermophilic. Preferred mesophilic DNA polymerases include T7DNA polymerase, T5 DNA polymerase, Klenow fragment DNA polymerase, DNApolymerase III and the like. Preferred thermostable DNA polymerases thatmay be used in the methods of the invention include Taq, Tne, Tma, Pfu,Tfl, Tth, Stoffel fragment, VENT™ and DEEPVENT™ DNA polymerases, andmutants, variants and derivatives thereof (U.S. Pat. No. 5,436,149; U.S.Pat. No. 4,889,818; U.S. Pat. No. 4,965,188; U.S. Pat. No. 5,079,352;U.S. Pat. No. 5,614,365; U.S. Pat. No. 5,374,553; U.S. Pat. No.5,270,179; U.S. Pat. No. 5,047,342; U.S. Pat. No. 5,512,462; WO92/06188; WO 92/06200; WO 96/10640; Barnes, W. M., Gene 112:29-35(1992); Lawyer, F. C., et al., PCR Meth. Appl. 2:275-287 (1993); Flaman,J.-M, et al., Nucl. Acids Res. 22(15):3259-3260 (1994)). Foramplification of long nucleic acid molecules (e.g., nucleic acidmolecules longer than about 3-5 Kb in length), at least two DNApolymerases (one substantially lacking 3′ exonuclease activity and theother having 3′ exonuclease activity) are typically used. See U.S. Pat.No. 5,436,149; and U.S. Pat. No. 5,512,462; Barnes, W. M., Gene112:29-35 (1992), the disclosures of which are incorporated herein intheir entireties. Examples of DNA polymerases substantially lacking in3′ exonuclease activity include, but are not limited to, Tag, Tne(exo⁻),Tma(exo⁻), Pfu (exo⁻), Pwo(exo⁻) and Tth DNA polymerases, and mutants,variants and derivatives thereof.

DNA polymerases for use in the present invention may be obtainedcommercially, for example, from Life Technologies, Inc. (Rockville,Md.), Pharmacia (Piscataway, N.J.), Sigma (St. Louis, Mo.) andBoehringer Mannheim. Preferred DNA polymerases for use in the presentinvention include Tsp DNA polymerase from Life Technologies, Inc.

Enzymes for use in the compositions, methods and kits of the inventioninclude any enzyme having reverse transcriptase activity. Such enzymesinclude, but are not limited to, retroviral reverse transcriptase,retrotransposon reverse transcriptase, hepatitis B reversetranscriptase, cauliflower mosaic virus reverse transcriptase, bacterialreverse transcriptase, Tth DNA polymerase, Taq DNA polymerase (Saiki, R.K., et al., Science 239:487-491 (1988); U.S. Pat. Nos. 4,889,818 and4,965,188), Tne DNA polymerase (WO 96/10640), Tma a DNA polymerase (U.S.Pat. No. 5,374,553) and mutants, fragments, variants or derivativesthereof (see, e.g., commonly owned, co-pending U.S. patent applicationSer. Nos. 08/706,702 and 08/706,706, both filed Sep. 9, 1996, which areincorporated by reference herein in their entireties). As will beunderstood by one of ordinary skill in the art, modified reversetranscriptases and DNA polymerase having RT activity may be obtained byrecombinant or genetic engineering techniques that are well-known in theart. Mutant reverse transcriptases or polymerases can, for example, beobtained by mutating the gene or genes encoding the reversetranscriptase or polymerase of interest by site-directed or randommutagenesis. Such mutations may include point mutations, deletionmutations and insertional mutations. Preferably, one or more pointmutations (e.g., substitution of one or more amino acids with one ormore different amino acids) are used to construct mutant reversetranscriptases or polymerases for use in the invention. Fragments ofreverse transcriptases or polymerases may also be obtained by deletionmutation by recombinant techniques that are well-known in the art, or byenzymatic digestion of the reverse transcriptase(s) or polymerase(s) ofinterest using any of a number of well-known proteolytic enzymes.

Preferred enzymes for use in the invention include those that arereduced or substantially reduced in RNase H activity. Such enzymes thatare reduced or substantially reduced in RNase H activity may be obtainedby mutating the RNase H domain within the reverse transcriptase ofinterest, preferably by one or more point mutations, one or moredeletion mutations, and/or one or more insertion mutations as describedabove. By an enzyme “substantially reduced in RNase H activity” is meantthat the enzyme has less than about 30%, less than about 25%, less thanabout 20%, more preferably less than about 15%, less than about 10%,less than about 7.5%, or less than about 5%, and most preferably lessthan about 5% or less than about 2%, of the RNase H activity of thecorresponding wildtype or RNase H⁺ enzyme such as wildtype MoloneyMurine Leukemia Virus (M-MLV), Avian Myeloblastosis Virus (AMV) or RousSarcoma Virus (RSV) reverse transcriptases. The RNase H activity of anyenzyme may be determined by a variety of assays, such as thosedescribed, for example, in U.S. Pat. No. 5,244,797, in Kotewicz, M. L.,et al., Nucl. Acids Res. 16:265 (1988), in Gerard, G. F., et al., FOCUS14(5):91 (1992), and in U.S. Pat. No. 5,668,005, the disclosures of allof which are fully incorporated herein by reference.

Polypeptides having reverse transcriptase activity for use in theinvention may be obtained commercially, for example from LifeTechnologies, Inc. (Rockville, Md.), Pharmacia (Piscataway, N.J.), Sigma(Saint Louis, Mo.) or Boehringer Mannheim Biochemicals (Indianapolis,Ind.). Alternatively, polypeptides having reverse transcriptase activitymay be isolated from their natural viral or bacterial sources accordingto standard procedures for isolating and purifying natural proteins thatare well-known to one of ordinary skill in the art (see, e.g., Houts. G.E., et al., J. Virol. 29:517 (1979)). In addition, the polypeptideshaving reverse transcriptase activity may be prepared by recombinant DNAtechniques that are familiar to one of ordinary skill in the art (see,e.g., Kotewicz, M. L., et al., Nucl. Acids Res. 16:265 (1988); Soltis,D. A., and Skalka, A. M., Proc. Natl. Acad. Sci. USA 85:3372-3376(1988)).

Preferred polypeptides having reverse transcriptase activity for use inthe invention include M-MLV reverse transcriptase, RSV reversetranscriptase, AMV reverse transcriptase, Rous Associated Virus (RAV)reverse transcriptase, Myeloblastosis Associated Virus (MAV) reversetranscriptase and Human Immunodeficiency Virus (HIV) reversetranscriptase, and others described in WO 98/47921 and derivatives,variants, fragments or mutants thereof, and combinations thereof. In afurther preferred embodiment, the reverse transcriptases are reduced orsubstantially reduced in RNase activity, and are most preferablyselected from the group consisting of M-MLV H⁻ reverse transcriptase,RSV H⁻ if reverse transcriptase, AMV H⁻ reverse transcriptase, RAV H⁻reverse transcriptase, MAV H⁻ reverse transcriptase and HIV H⁻ reversetranscriptase, and derivatives, variants, fragments or mutants thereof,and combinations thereof. Reverse transcriptases of particular interestinclude AMV RT and M-MLV RT, and more preferably AMV RT and M-MLV RThaving reduced or substantially reduced RNase H activity (preferably AMVRT αH⁻/BH⁻ and M-MLV RT H⁻) The most preferred reverse transcriptasesfor use in the invention include Super Script™, SuperScript™ II,ThermoScript™ and ThermoScript™ II available from Life Technologies,Inc. See generally, WO 98/47921, U.S. Pat. Nos. 5,244,797 and 5,668,005,the entire contents of each of which are herein incorporated byreference.

Hairpin. As used herein, the term “hairpin” is used to indicate thestructure of an oligonucleotide in which one or more portions of theoligonucleotide form base pairs with one or more other portions of theoligonucleotide. When the two portions are base paired to form a doublestranded portion of the oligonucleotide, the double stranded portion maybe referred to as a stem. Thus, depending on the number of complementaryportions used, a number of stems (preferably 1-10) may be formed.Additionally, formation of the one or more stems preferably allowsformation of one or more loop structures in the hairpin molecule. In oneaspect, any one or more of the loop structures may be cut or nicked atone or more sites within the loop or loops but preferably at least oneloop is not so cut or nicked. The sequence of the oligonucleotide may beselected so as to vary the number of nucleotides which base pair to formthe stem from about 3 nucleotides to about 100 or more nucleotides, fromabout 3 nucleotides to about 50 nucleotides, from about 3 nucleotides toabout 25 nucleotides, and from about 3 to about 10 nucleotides. Inaddition, the sequence of the oligonucleotide may be varied so as tovary the number of nucleotides which do not form base pairs from 0nucleotides to about 100 or more nucleotides, from 0 nucleotides toabout 50 nucleotides, from 0 nucleotides to about 25 nucleotides or from0 to about 10 nucleotides. The two portions of the oligonucleotide whichbase pair may be located anywhere or at any number of locations in thesequence of the oligonucleotide. In some embodiments, onebase-pairing-portion of the oligonucleotide may include the 3′-terminalof the oligonucleotide. In some embodiments, one base-pairing-portionmay include the 5′-terminal of the oligonucleotide. In some embodiments,one base-pairing-portion of the oligonucleotide may include the3′-terminal while the other base-pairing-portion may include the5′-terminal and, when base paired, the stem of the oligonucleotide isblunt ended. In other embodiments, the location of the base pairingportions of the oligonucleotide may be selected so as to form a3′-overhang, a 5′-overhang and/or may be selected so that neither the3′-nor the 5′-most nucleotides are involved in base pairing.

Hybridization. As used herein, the terms “hybridization” and“hybridizing” refer to the pairing of two complementary single-strandednucleic acid molecules (RNA and/or DNA) to give a double-strandedmolecule. As used herein, two nucleic acid molecules may be hybridized,although the base pairing is not completely complementary. Accordingly,mismatched bases do not prevent hybridization of two nucleic acidmolecules provided that appropriate conditions, well known in the art,are used.

Incorporating. The term “incorporating” as used herein means becoming apart of a DNA or RNA molecule or primer.

Nucleotide. As used herein “nucleotide” refers to a base-sugar-phosphatecombination. Nucleotides are monomeric units of a nucleic acid sequence(DNA and RNA). The term nucleotide includes mono-, di- and triphosphateforms of deoxyribonucleosides and ribonucleosides and their derivatives.The term nucleotide particularly includes deoxyribonucleosidetriphosphates such as dATP, dCTP, dITP, dUTP, dGTP, dTTP, or derivativesthereof. Such derivatives include, for example, [αS]dATP, 7-deaza-dGTPand 7-deaza-dATP. The term nucleotide as used herein also refers todideoxyribonucleoside triphosphates (ddNTPs) and their derivatives.Illustrated examples of dideoxyribonucleoside triphosphates include, butare not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP. According tothe present invention, a “nucleotide” may be unlabeled or detectablylabeled by well known techniques. Detectable labels include, forexample, radioactive isotopes, fluorescent labels, chemiluminescentlabels, bioluminescent labels and enzyme labels.

Oligonucleotide. As used herein, “oligonucleotide” refers to a syntheticor biologically produced molecule comprising a covalently linkedsequence of nucleotides which may be joined by a phosphodiester bondbetween the 3′ position of the pentose of one nucleotide and the 5′position of the pentose of the adjacent nucleotide. Oligonucleotide asused herein is seen to include natural nucleic acid molecules (i.e., DNAand RNA) as well as non-natural or derivative molecules such as peptidenucleic acids, phophothioate containing nucleic acids, phosphonatecontaining nucleic acids and the like. In addition, oligonucleotides ofthe present invention may contain modified or non-naturally occurringsugar residues (i.e., arabainose) and/or modified base residues.Oligonucleotide is seen to encompass derivative molecules such asnucleic acid molecules comprising various natural nucleotides,derivative nucleotides, modified nucleotides or combinations thereof.Thus any oligonucleotide or other molecule useful in the methods of theinvention are contemplated by this definition. Oligonucleotides of thepresent invention may also comprise blocking groups which prevent theinteraction of the molecule with particular proteins, enzymes orsubstrates.

Primer. As used herein, “primer” refers to a synthetic or biologicallyproduced single-stranded oligonucleotide that is extended by covalentbonding of nucleotide monomers during amplification or polymerization ofa nucleic acid molecule. Nucleic acid amplification often is based onnucleic acid synthesis by a nucleic acid polymerase or reversetranscriptase. Many such polymerases or reverse transcriptases requirethe presence of a primer that can be extended to initiate such nucleicacid synthesis. A primer is typically 11 bases or longer; mostpreferably, a primer is 17 bases or longer, although shorter or longerprimers may be used depending on the need. As will be appreciated bythose skilled in the art, the oligonucleotides of the invention may beused as one or more primers in various extension, synthesis oramplification reactions.

Probe. As used herein, “probe” refers to synthetic or biologicallyproduced nucleic acids (DNA or RNA) which, by design or selection,contain specific nucleotide sequences that allow them to hybridize,under defined stringencies, specifically (i.e., preferentially) totarget nucleic acid sequences. As will be appreciated by those skilledin the art, the oligonucleotides of the present invention may be used asone or more probes and preferably may be used as probes for thedetection or quantification of nucleic acid molecules. Substantiallyless extendable. As used herein, “substantially less extendable” is usedto characterize an oligonucleotide that is inefficiently extended or notextended in an extension and/or amplification reaction when the 3′-mostnucleotide of the oligonucleotide is not complementary to thecorresponding base of a target/template nucleic acid. Preferably, anoligonucleotide is substantially less extendable as a result of thepresence of a specificity enhancing group on the oligonucleotide. Inthis event, an oligonucleotide is substantially less extendable when theoligonucleotide is not extended or is extended by a lesser amount and/orat a slower rate than an oligonucleotide lacking the specificityenhancing group but having an otherwise identical structure. Thoseskilled in the art can readily determine if an oligonucleotide issubstantially less extendable by conducting an extension reaction usingan oligonucleotide containing a specificity enhancing group andcomparing the extension to the extension of an oligonucleotide of thesame structure but lacking the specificity enhancing group. Underidentical extension conditions, (e.g., melting temperature and time,annealing temperature and time, extension temperature and time, reactantconcentrations and the like), a substantially less extendableoligonucleotide will produce less extension product when the 3′-mostnucleotide of the oligonucleotide is not complementary to thecorresponding nucleotide on a target/template nucleic acid than will beproduced by an oligonucleotide lacking a specificity enhancing group buthaving an otherwise identical structure. Alternatively, one skilled inthe art can determine if an oligonucleotide is substantially lessextendable by conducting allele specific PCR with a first set ofoligonucleotides at least one of which comprises one or more specificityenhancing groups and with a second set of oligonucleotides lackingspecificity enhancing groups but otherwise of identical structure tothose of the first set. Then a determination is separately made for eachset of primers of the difference in the amount of product made and/orthe rate at which the product is made with the oligonucleotide havingthe 3′-nucleotide complementary to the corresponding nucleotide on atarget/template nucleic acid to the amount of product made and/or therate at which the product is made with an oligonucleotide having the3′-nucleotide not complementary to the corresponding nucleotide on atarget/template nucleic acid. Substantially less extendableoligonucleotides will produce a larger difference in amount of productmade and/or rate at which product is made between 3′-complementary and3′-not-complementary oligonucleotides. Preferably the difference in theamount of product made and/or rate at which product is made usingoligonucleotides containing specificity enhancing groups will be betweenfrom about 1.1 fold to about 1000 fold larger than the differenceobtained using primers lacking specificity enhancing groups, or fromabout 1.1 fold to about 500 fold larger, or from about 1.1 fold to about250 fold larger, or from about 1.1 fold to about 100 fold larger, orfrom about 1.1 fold to about 50 fold larger, or from about 1.1 to about25 fold larger, or from about 1.1 to about 10 fold larger, or from about1.1 fold to about 5 fold or from about 1.1 fold to about 2 fold larger.The amount of product can be determined using any methodology known tothose of skill in the art, for example, by running the product on anagarose gel and staining with ethidium bromide and comparing to knownamounts of similarly treated nucleic acid standards. The amount ofproduct may be determined at any convenient time point in the allelespecific PCR. One convenient way to compare the rate of formation ofproduct is to compare the number of cycles required to form a specifiedamount of product in a PCR. A determination is separately made for eachset of primers of the difference between the number of cycles requiredto make a given amount of product with the oligonucleotide having the3′-nucleotide complementary to the corresponding nucleotide on atarget/template nucleic acid and the number of cycles required to makethe same amount of product with an oligonucleotide having the3′-nucleotide not complementary to the corresponding nucleotide on atarget/template nucleic acid. Substantially less extendableoligonucleotides will produce a larger difference in the number ofcycles required to produce a specified amount of product between3′-complementary and 3′-not-complementary oligonucleotides. The amountof product made can be determined using any means known to those skilledin the art, for example, by determining the fluorescence intensity of alabeled product using a thermocycler adapted to perform real timefluorescence detection. Preferably the difference between the number ofcycles required to make a specified amount of product usingoligonucleotides containing specificity enhancing groups will be betweenfrom about 1.05 fold to about 100 fold larger than the differenceobtained using primers lacking specificity enhancing groups, or fromabout 1.05 fold to about 50 fold larger, or from about 1.05 fold toabout 25 fold larger, or from about 1.05 fold to about 10 fold larger,or from about 1.05 fold to about 5 fold larger, or from about 1.05 toabout 2.5 fold larger, or from about 1.05 to about 1.5 fold larger, orfrom about 1.05 fold to about 1.2 fold larger.

Support. As used herein a “support” may be any material or matrixsuitable for attaching the oligonucleotides of the present invention ortarget/template nucleic acid sequences. Such oligonucleotides and/orsequences may be added or bound (covalently or non-covalently) to thesupports of the invention by any technique or any combination oftechniques well known in the art. Supports of the invention may comprisenitrocellulose, diazocellulose, glass, polystrene (including microtitreplates), polyvinylchloride, polypropylene, polyethylene, dextran,Sepharose, agar, starch and nylon. Supports of the invention may be inany form or configuration including beads, filters, membranes, sheets,frits, plugs, columns and the like. Solid supports may also includemulti-well tubes (such as microtitre plates) such as 12-well plates,24-well plates, 48-well plates, 96-well plates, and 384-well plates.Preferred beads are made of glass, latex or a magnetic material(magnetic, paramagnetic or superparamagnetic beads).

In a preferred aspect, methods of the invention may be used inconjunction with arrays of nucleic acid molecules (RNA or DNA). Arraysof nucleic acid template/target or arrays of oligonucleotides of theinvention are both contemplated in the methods of the invention. Sucharrays may be formed on microplates, glass slides or standard blottingmembranes and may be referred to as microarrays or gene-chips dependingon the format and design of the array. Uses for such arrays include genediscovery, gene expression profiling and genotyping (SNP analysis,pharmacogenomics, toxicogenetics).

Synthesis and use of nucleic acid arrays and generally attachment ofnucleic acids to supports have been described (see for example, U.S.Pat. No. 5,436,327, U.S. Pat. No. 5,800,992, U.S. Pat. No. 5,445,934,U.S. Pat. No. 5,763,170, U.S. Pat. No. 5,599,695 and U.S. Pat. No.5,837,832). An automated process for attaching various reagents topositionally defined sites on a substrate is provided in Pirrung et al.U.S. Pat. No. 5,143,854 and Barrett et al. U.S. Pat. No. 5,252,743.

Essentially, any conceivable support may be employed in the invention.The support may be biological, nonbiological, organic, inorganic, or acombination of any of these, existing as particles, strands,precipitates, gels, sheets, tubing, spheres, containers, capillaries,pads, slices, films, plates, slides, etc. The support may have anyconvenient shape, such as a disc, square, sphere, circle, etc. Thesupport is preferably flat but may take on a variety of alternativesurface configurations. For example, the support may contain raised ordepressed regions on which one or more methods of the invention may takeplace. The support and its surface preferably form a rigid support onwhich to carry out the reactions described herein. The support and itssurface is also chosen to provide appropriate light-absorbingcharacteristics. For instance, the support may be a polymerized LangmuirBlodgett film, functionalized glass, Si, Ge, GaAs, GaP, SiO₂, SIN₄,modified silicon, or any one of a wide variety of gels or polymers suchas (poly)tetrafluoroethylene, (poly)vinylidenedifluoride, polystyrene,polycarbonate, or combinations thereof. Other support materials will bereadily apparent to those of skill in the art upon review of thisdisclosure. In a preferred embodiment the support is flat glass orsingle-crystal silicon.

Target molecule. As used herein, “target molecule” refers to a nucleicacid molecule to which a particular primer or probe is capable ofpreferentially hybridizing.

Target sequence. As used herein, “target sequence” refers to a nucleicacid sequence within the target molecules to which a particular primeror probe is capable of preferentially hybridizing.

Template. The term “template” as used herein refers to a double-strandedor single-stranded molecule which is to be amplified, synthesized orsequenced. In the case of a double-stranded DNA molecule, denaturationof its strands to form a first and a second strand is preferablyperformed to amplify, sequence or synthesize these molecules. A primer,complementary to a portion of a template is hybridized under appropriateconditions and the polymerase (DNA polymerase or reverse transcriptase)may then synthesize a nucleic acid molecule complementary to saidtemplate or a portion thereof. The newly synthesized molecule, accordingto the invention, may be equal or shorter in length than the originaltemplate. Mismatch incorporation during the synthesis or extension ofthe newly synthesized molecule may result in one or a number ofmismatched base pairs. Thus, the synthesized molecule need not beexactly complementary to the template. The template can be an RNAmolecule, a DNA molecule or an RNA/DNA hybrid molecule. A newlysynthesized molecule may serve as a template for subsequent nucleic acidsynthesis or amplification.

Thermostable. As used herein “thermostable” refers to a polymerase (RNA,DNA or RT) which is resistant to inactivation by heat. DNA polymerasessynthesize the formation of a DNA molecule complementary to asingle-stranded DNA template by extending a primer in the 5′-to-3′direction. This activity for mesophilic DNA polymerases may beinactivated by heat treatment. For example, T5 DNA polymerase activityis totally inactivated by exposing the enzyme to a temperature of 90° C.for 30 seconds. As used herein, a thermostable DNA polymerase activityis more resistant to heat inactivation than a mesophilic DNA polymerase.However, a thermostable DNA polymerase does not mean to refer to anenzyme which is totally resistant to heat inactivation and thus heattreatment may reduce the DNA polymerase activity to some extent. Athermostable DNA polymerase typically will also have a higher optimumtemperature than mesophilic DNA polymerases.

Other terms used in the fields of recombinant DNA technology andmolecular and cell biology as used herein will be generally understoodby one of ordinary skill in the applicable arts.

The present invention provides oligonucleotides, which may be labeledinternally, and/or, at or near the 3′ termini and/or 5′ termini or maybe unlabeled. In another aspect, the oligonucleotides of the presentinvention may be provided with a specificity enhancing group. Such agroup may be located internally and/or at or near the 3′- and/or the5′-terminal of the oligonucleotide. In another aspect, theoligonucleotides of the present invention may be in the form of ahairpin. In some preferred embodiments, the oligonucleotides may beprovided with more than one of these characteristics, i.e., they maycomprise a label and/or a specificity enhancing group and/or may be inthe form of a hairpin.

When labeled, oligonucleotides of the invention may contain one ormultiple labels (which may be the same or different). Theoligonucleotides of the invention may be used as primers and/or probes.In a preferred aspect, the oligonucleotides are labeled and the label isany moiety which undergoes a detectable change in any observableproperty upon hybridization and/or extension. In a preferredembodiments, the label is a fluorescent moiety and the label undergoes adetectable change in one or more fluorescent properties. Such propertiesare seen to include, but are not limited to, fluorescent intensity,fluorescent polarization, fluorescent lifetime and quantum yield offluorescence. The oligonucleotides for use in the invention can be anysuitable size, and are preferably in the range of 10-100 or 10-80nucleotides, more preferably 11-40 nucleotides and most preferably inthe range of 17-25 nucleotides although oligonucleotides may be longeror shorter depending upon the need.

The oligonucleotides of the invention can be DNA or RNA or chimericmixtures or derivatives or modified versions thereof. In addition tobeing labeled with a detectable moiety, the oligonucleotide can bemodified at the base moiety, sugar moiety, or phosphate backbone, andmay include other appending groups or labels.

For example, the oligonucleotides of the invention may comprise at leastone modified or more base moieties which are selected from the groupincluding but not limited to 5-fluorouracil, 5-bromouracil,5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine,5-(carboxyhydroxylmethyl) uracil,5-carboxymethylamino-methyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N⁶-isopentenyladenine,1-methylguanine, 1-methyl-linosine, 2,2-dimethylguanine,2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine,N⁶-adenine, 7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxy-methyluracil, 5-methoxyuracil,2-methylthio-N⁶-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine.

In another embodiment, the oligonucleotides of the invention comprisesat least one modified sugar moiety selected from the group including butnot limited to arabinose, 2-fluoroarabinose, xylulose, and hexose.

In yet another embodiment, the oligonucleotides of the inventioncomprises at least one modified phosphate backbone selected from thegroup consisting of a phosphorothioate, a phosphorodithioate, aphosphoramidothioate, a phosphoramidate, a phosphordiamidate, amethylphosphonate, an alkyl phosphotriester, and a formacetal or analogthereof.

The oligonucleotides of the invention have use in nucleic acidamplification or synthesis reactions (e.g., as primers) to detect ormeasure a nucleic acid product of the amplification or synthesisreaction, thereby detecting or measuring a target nucleic acid in asample that is complementary to all or a portion of a primer sequence.The oligonucleotides of the invention may be used in any amplificationreactions including PCR, 5-RACE, Anchor PCR, “one-sided PCR,” LCR,NASBA, SDA, RT-PCR and other amplification systems known in the art.

Thus, the invention generally relates to methods of synthesizing oramplifying one or more nucleic acid molecules comprising:

-   (a) mixing one or more templates or target nucleic acid molecules    with one or more oligonucleotides of the invention; and-   (b) incubating said mixture under conditions sufficient to    synthesize or amplify one or more nucleic acid molecules    complementary to all or a portion of said templates or target    molecules.    Preferably, the synthesized or amplified nucleic acid molecules    comprise one or more oligonucleotides of the invention or portions    thereof. In one aspect, the oligonucleotides of the invention are    incorporated at or near one or both termini of the synthesized or    amplified nucleic acid molecules produced by the methods of the    invention. The invention also relates to one or more nucleic acid    molecules produced by such amplification or synthesis reactions.

In another aspect, the invention relates to methods of synthesizing oneor more nucleic acid molecules, comprising

-   (a) mixing one or more nucleic acid templates (which may be DNA    molecules such as a cDNA molecules, or RNA molecules such as mRNA    molecules, or populations of such molecules) with one or more    primers of the invention and one or more polymerases; and-   (b) incubating the mixture under conditions sufficient to synthesize    one or more first nucleic acid molecules complementary to all or a    portion of the templates.    Such incubation conditions may involve the use of one or more    nucleotides and one or more nucleic acid synthesis buffers. Such    methods of the invention may optionally comprise one or more    additional steps, such as incubating the synthesized first nucleic    acid molecules under conditions sufficient to make one or more    second nucleic acid molecules complementary to all or a portion of    the first nucleic acid molecules. Such additional steps may also be    accomplished in the presence of one or more primers of the invention    and one or more polymerases as described herein. The invention also    relates to nucleic acid molecules synthesized by these methods.

The invention also relates to methods for sequencing nucleic acidmolecules comprising

-   (a) mixing a nucleic acid molecule to be sequenced with one or more    primers of the invention, one or more nucleotides and one or more    terminating agents to form a mixture;-   (b) incubating the mixture under conditions sufficient to synthesize    the population of molecules complementary to all or a portion of the    molecule to be sequence; and-   (c) separating the population to determining the nucleotide sequence    of all or a portion of the molecule to be sequenced.

The invention more specifically relates to a method of sequencing anucleic acid molecule, comprising:

-   (a) mixing one or more of the oligonucleotides of the invention, one    or more nucleotides, and one or more terminating agents;-   (b) hybridizing said oligonucleotides to a first nucleic acid    molecule;-   (c) incubating the mixture of step (b) under conditions sufficient    to synthesize a random population of nucleic acid molecules    complementary to said first nucleic acid molecule, wherein said    synthesized molecule are shorter in length than said first molecule    and wherein said synthesized molecules comprise a terminator    nucleotide at their 3′ termini; and

separating said synthesized molecules by size so that at least a part ofthe nucleotide sequence of said first nucleic acid molecule can bedetermined. Such terminator nucleotides include ddTTP, ddATP, ddGTP,ddITP or ddCTP. Such incubation conditions may include incubation in thepresence of one or more polymerases and/or buffering salts.

In a related aspect, the oligonucleotides of the invention are useful indetecting the presence or absence of or quantifying the amount ofnucleic acid molecules in a sample without the need for performingamplification or synthesis reactions. In accordance with the invention,an oligonucleotide may be provided with one or more labels which undergoa detectable change in at least one observable property when theoligonucleotide comprising the label is converted to a double strandedmolecule (e.g., by hybridizing the oligonucleotide to a targetmolecule). Thus, a change in an observable property indicates thepresence of the target molecule in the sample when compared to a controlsample not containing the nucleic acid molecule of interest.Quantification of the nucleic acid target molecule in the sample mayalso be determined by comparing change in the observable property in anunknown sample to the changes in the observable property in samplescontaining known amounts of the nucleic acid target molecule ofinterest. Any samples thought to contain the nucleic acid molecule ofinterest may be used including, but not limited to, biological samplessuch as blood, urine, tissue, cells, feces, serum, plasma, or any othersamples derived from animals (including humans), plants, bacteria,viruses and the like. Environmental samples such as soil samples, watersamples, air samples and the like may also be used in accordance withthe invention.

The oligonucleotides of the invention can be used in methods ofdiagnosis, wherein the oligonucleotide is complementary to a sequence(e.g., genomic or cDNA) of an infectious disease agent or is capable ofinitiating synthesis or amplification of a sequence of an infectiousdisease agent, e.g. of human disease including but not limited toviruses (e.g, HIV, HPV etc), bacteria, parasites, and fungi, therebydiagnosing the presence of the infectious agent in a sample from apatient. The type of target nucleic acid can be genomic, cDNA, mRNA,synthetic, or the source may be human, animal, or bacterial. In anotherembodiment that can be used in the diagnosis or prognosis of a diseaseor disorder, the target sequence is a wild type human genomic or RNA orcDNA sequence, mutation of which is implicated in the presence of ahuman disease or disorder, or alternatively, can be the mutatedsequence. In such an embodiment, the hybridization, amplification orsynthesis reaction of the invention can be repeated for the same samplewith different sets of oligonucleotides of the invention (for example,with differently labeled oligonucleotide) which selectively identify thewild type sequence or the mutated version. By way of example, themutation can be an insertion, substitution, and/or deletion of one ormore nucleotides, or a translocation.

In a specific embodiment, the invention provides a method for detectingor measuring a product of a nucleic acid amplification or synthesisreaction comprising (a) contacting a sample comprising one or moretarget nucleic acid molecules with one or more primers (such primers maycomprise one or multiple labels, which may be the same or different andmay be labeled internally, and/or, at or near the 3′ and/or 5′ end),said primers being adapted for use in said amplification or synthesisreaction such that said primers are incorporated into an amplified orsynthesized product of said amplification or synthesis reaction when atarget sequence or nucleic acid molecule is present in the sample; (b)conducting the amplification or synthesis reaction; and (c) detecting ormeasuring one or more synthesis or amplification product molecules(preferably by detecting a change in one or more observable propertiesof one or more labels).

In another specific embodiment, the invention provides for a method ofdetecting or measuring the presence or absence or the amount of a targetnucleic acid molecule within a sample comprising (a) contacting a samplecomprising one or more target nucleic acid molecules with one or moreoligonucleotides of the invention (such oligonucleotides may compriseone or multiple labels, which may be the same or different and may belabeled internally and/or at or near the 3′ and/or 5′ end); (b)incubating said mixture under conditions sufficient to allow saidoligonucleotides to interact with said target molecules sufficient toform double stranded molecules (preferably through hybridization); and(c) detecting one or more of said target nucleic acid molecules(preferably by detecting a change in one or more observable propertiesof one or more labels).

The present invention provides a method for detecting a target nucleicacid sequence, comprising the steps of contacting a sample containing amixture of nucleic acids with at least one oligonucleotide of thepresent invention, the oligonucleotide capable of hybridizing a targetnucleic acid sequence and comprises at least one detectable moiety,wherein the detectable moiety undergoes a change in one or moreobservable properties upon hybridization to the target nucleic acidsequence and observing the observable property, wherein a change in theobservable property indicates the presence of the target nucleic acidsequence. In some embodiments, the target nucleic acid sequence is notseparated from the mixture. In some embodiments, the observable propertyis fluorescence. In some embodiments, the change is an increase influorescence. In some embodiments, the change is a decrease influorescence. In some embodiments, the oligonucleotide comprises aspecificity enhancing group. In some embodiments, the oligonucleotide isin the form of a hairpin.

The present invention provides a method for quantifying a target nucleicacid molecule, comprising the steps contacting a sample containing amixture of nucleic acids comprising the target nucleic acid moleculewith at least one oligonucleotide of the present invention, theoligonucleotide capable 15 of hybridizing to the target nucleic acidmolecule and comprises at least one detectable moiety, wherein thedetectable moiety undergoes a change in one or more observableproperties upon hybridization to the target nucleic acid sequence andobserving the observable property, wherein a change in the observableproperty is proportional to the amount of the target nucleic acidmolecule in the sample.

In a further aspect, the invention relates to the use of one or moretreatments to lower or decrease the energy emitted by the labels of theoligonucleotides of the invention. Such treatments may be used inaccordance with the invention to lower the background in thehybridization, synthesis or amplification methods of the invention. Inone aspect, single stranded nucleic acid binding protein (E. coli, T4bacteriophage or Archaea (see Kelly, et al. Proceedings of the NationalAcademy of Sciences, USA 95:14634-14639 (1998), Chedin, et al., TIBS23:273-277 (1998), U.S. Pat. Nos. 5,449,603, 5,605,824, 5,646,019, and5,773,257) may be used to interact with single stranded labeledoligonucleotides of the invention to reduce or quench energy emitted orother detectable properties from the labels. Such single strandedbinding proteins may be native or modified. During the detection orquantitation process (hybridization, synthesis or amplificationreactions) double stranded nucleic acid molecules formed do notsubstantially interact with single stranded binding protein or interactminimally with such double stranded molecules. Accordingly, in theunreacted state (single stranded form of the oligonucleotides of theinvention), energy emitted or other detectable properties (e.g.,fluorescence) is reduced or quenched while in the reactive form (doublestranded molecules) energy emitted or other detectable properties isenhanced. In another aspect, blocking oligonucleotides which containquencher molecules may be used to competitively bind the labeledoligonucleotides in the invention in the unreacted stated therebyreducing energy emitted or other detectable properties of the labeledoligonucleotide. In another aspect, one or more additional fluorescentmoieties may be incorporated into the blocking molecule such that thefluorescent moiety on the oligonucleotide of the invention is inproximity to the one or more additional fluorescent moieties when theoligonucleotide of the invention is in the unreacted state. The presenceof an additional fluorescent molecule can reduce the backgroundfluorescence level even though there is little or no overlap between theemission spectrum of the fluorescent moiety on the oligonucleotide ofthe invention and the absorption spectrum of the one or more additionalfluorescent moieties on the blocking oligonucleotide. When theoligonucleotide of the invention has the capability of forming a hairpinstructure, those skilled in the art will appreciate the one or moreadditional fluorescent moieties can be brought into proximity with thelabel on the oligonucleotide of the invention by attaching the one ormore additional fluorescent moieties to nucleotides in one strand of thestem structure of the hairpin while attaching one or more labels tonucleotides in the other strand. During detection or quantitation,target nucleic acid molecules interact with labeled oligonucleotides ofthe invention thereby enhancing energy emitted or other detectableproperties by the labels. Such interaction may separate the blockingoligonucleotide (e.g., quencher/additional fluorescent moiety-containingmolecule) from the label containing oligonucleotide of the invention.

In another aspect of the present invention, the sequence of theoligonucleotide and/or a blocking oligonucleotide may be selected so asto reduce the background fluorescence of the oligonucleotides of theinvention. It has been unexpectedly found that the base sequence in thevicinity of the label can have a dramatic effect on the backgroundfluorescence level. The background fluorescence of a single strandedoligonucleotide of the present invention can be decreased about 5 foldif the sequence of the oligonucleotide is selected so as to form ablunt-end double stranded structure with one or more fluorophoreslocated on one or more base close to the 3′-end and G-C or C-G base pairbeing the last base pair of the double stranded structure. In somepreferred embodiments, the double stranded structure may be a stem of ahairpin structure. In some preferred embodiments, the 3′-end of theoligonucleotides of the invention may be provide with one of thefollowing sequences: 5′- . . . T(Fluo)C-3′, 5′- . . . T(Fluo)G-3′, 5′- .. . T(Fluo)AG-3′, 5′- . . . T(Fluo)AC-3′, 5′- . . . T(Fluo)TC-3′, 5′- .. . T(Fluo)TG-3′ where the attachment of a fluorophore is indicated by(Fluo) and the 3′-sequence is as shown while the blockingoligonucleotide (or 5′-end of a hairpin oligonucleotide) is providedwith the complementary sequence (preferably at the 5′ end of theblocking oligonucleotide/hairpin molecule). To achieve a quenchingeffect the labeled base should be within 10 nucleotides distance fromthe 3′-end, preferably within 6 nucleotides and most preferably within1-4 nucleotides. A specific example of oligonucleotides of this type isprovided by Oligo 10 (SEQ ID NO:22) in Table 2. In a related embodiment,when using an oligonucleotide that does not have G or C for its 3′-mostnucleotide and hence cannot form a G-C base pair at the 3′-end, theaddition of a 5′-overhanging G residue to the oligonucleotide can reducethe background fluorescence. Also, the presented mode of quenching canbe combined with the another mechanism of quenching like fluorescenceresonance energy transfer or static quenching. In some embodiments ofthe present invention, combinations of quenching techniques may beemployed to reduce the background fluorescence. For example, anoligonucleotide of the present invention may have a detectable moietylocated near the 3′ end of the oligonucleotide while the sequence of theoligonucleotide may be selected so as to have a G-C base pair at a bluntend of a hairpin structure and one or more additional fluorescentmoieties may be attached to nucleotides at or near the 5′-end of theoligonucleotide. A similar structure could be employed utilizing ablocking oligonucleotide instead of a hairpin.

Other means for quenching or reducing nonreacted labeledoligonucleotidcs may be used or any combination of such treatments maybe used in accordance with the invention.

The present invention provides a composition comprising one or moreoligonucleotides of the invention and one or more target or templatenucleic acid molecules, wherein at least a portion of theoligonucleotide is capable of hybridizing to at least a portion of thetarget or template nucleic acid molecule (preferably the oligonucleotidecomprises one or more detectable moieties that undergo a change in oneor more observable property upon hybridization to the target nucleicacid molecule). In some embodiments, the detectable moiety is afluorescent moiety and the fluorescent moiety undergoes a change influorescence upon hybridizing to the target nucleic acid molecule. Insome embodiments, the oligonucleotide is a hairpin when not hybridizedto the target nucleic acid molecule.

In some preferred embodiments, the present invention provides acomposition comprising at least one nucleic acid molecule and at leastone oligonucleotide of the invention, wherein at least a portion of saidoligonucleotide is capable of hybridizing with at least a portion ofsaid nucleic acid molecule and wherein said oligonucleotide comprisesone or more specificity enhancing groups. In some embodiments, one ormore of the specificity enhancing groups may be a fluorescent moiety. Aspecificity enhancing group may be attached at any position of theoligonucleotide that results in the oligonucleotide being substantiallyless extendable when the 3′most nucleotide of the oligonucleotide is notcomplementary to the corresponding nucleotide of a target/templatenucleic acid. In some embodiments, at least one of the one or moregroups is attached to a nucleotide at or near the 3′-nucleotide. In someembodiments, at least one of the one or more groups is attached to one,of the ten 3′-most nucleotides. In other words, in embodiments of thistype, at least one of the one or more specificity enhancing groups maybe attached to the 3′-most nucleotide or any of the next nine contiguousnucleotides in the 5′-direction. In some embodiments, at least one ofthe one or more groups is attached to one of the five 3′-mostnucleotides. In some embodiments, the group may be a label, preferably alabel which undergoes a detectable change in an observable property uponbecoming part of a double stranded molecule, (e.g. by hybridizing toanother nucleic acid molecule or by nucleic acid synthesis oramplification). In some embodiments, at least a portion of saidoligonucleotide is hybridized to at least a portion of said nucleic acidmolecule. In some embodiments, the oligonucleotide is capable of forminga hairpin. In some embodiments, the oligonucleotide is in the form of ahairpin.

The present invention provides a method of making a composition,comprising the steps of providing one or more oligonucleotides andcontacting the one or more oligonucleotides with at least one nucleicacid molecule, wherein at least a portion of at least one of saidoligonucleotides is capable of hybridizing with at least a portion ofsaid nucleic acid molecule. Preferably, the oligonucleotide comprisesone or more specificity enhancing groups and/or at least one detectablelabel. In some embodiments, the group is a fluorescent moiety. Aspecificity enhancing group may be attached at any position of theoligonucleotide that results in the oligonucleotide being substantiallyless extendable when the 3′-most nucleotide of the oligonucleotide isnot complementary to the corresponding nucleotide of a target/templatenucleic acid. In some embodiments, at least one of the one or moregroups is attached to a nucleotide at or near the 3′-nucleotide. In someembodiments, at least one of the one or more groups is attached to oneof the ten 3′-most nucleotides. In other words, in embodiments of thistype, at least one of the one or more specificity enhancing groups maybe attached to the 3′most nucleotide or any of the next nine contiguousnucleotides in the 5′direction. In some embodiments, at least one of theone or more groups is attached to one of the five 3′-most nucleotides.In some embodiments, the group may be a label, preferably a label whichundergoes a detectable change in an observable property upon becomingpart of a double stranded molecule, (e.g. by hybridizing to anothernucleic acid molecule). In some embodiments, at least a portion of saidoligonucleotide is hybridized to at least a portion of said nucleic acidmolecule. In some embodiments, the oligonucleotide is capable of forminga hairpin. In some embodiments, the oligonucleotide is in the form of ahairpin.

The present invention provides a method of determining the presence of aparticular nucleotide or nucleotides at a specific position or positionsin a target or template nucleic acid molecule, comprising the steps of(a) contacting at least one target or template nucleic acid moleculehaving a nucleotide or nucleotides at a specific position or positionswith one or more oligonucleotides of the invention, wherein at least aportion of the oligonucleotide is capable of forming base pairs (e.g.,hybridizing) with at least a portion of the target or template nucleicacid molecule said oligonucleotide preferably comprises at least onespecificity enhancing group and/or label; and (b) incubating theoligonucleotide and the nucleic acid molecule mixture under conditionssufficient to cause extension of the oligonucleotide when the 3′-mostnucleotide or nucleotides of the oligonucleotide base pair with thenucleotide or nucleotides at the specific position or positions of thenucleic acid target molecule. Under such conditions, the production ofan extension product indicates the presence of the particular nucleotideor nucleotides at the specific position or positions. In another aspect,the invention provides a method for determining the absence of at leastone particular nucleotide at a specific position or positions in atarget or template nucleic acid molecule, comprising (a) contacting atleast one target nucleic acid molecule having a nucleotide ornucleotides at a specific position with an oligonucleotide of theinvention, wherein at least a portion of the oligonucleotide is capableof forming base pairs (e.g., hybridizing) with at least a portion of thetarget nucleic acid molecule (said oligonucleotide preferably comprisingat least one specificity enhancing group or label); and (b) incubatingthe oligonucleotide and the nucleic acid molecule mixture underconditions sufficient to prevent or inhibit extension of theoligonucleotide when the 3′-most nucleotide or nucleotides of theoligonucleotide does not base pair (e.g., does not hybridize) with thenucleotide at the specific position or positions of the target nucleicacid molecule. Under such conditions, the lack of production or reducedproduction of an extension product indicates the absence of theparticular nucleotide or nucleotides at the specific position. In apreferred aspect, the results of the extension of the oligonucleotide inthe above first method is compared to the lack or reduced level ofextension of the oligonucleotide in the above second method. In apreferred aspect, the conditions in the first method are conducted suchthat all or a portion of the target nucleic acid molecule is amplified,while the conditions in the second method are conducted such that thetarget nucleic acid molecule is not amplified or amplified at a reducedlevel or slower rate compared to the amplified target nucleic acidmolecule produced by the first method. In some embodiments, thespecificity enhancing group is a fluorescent moiety. A specificityenhancing group may be attached at any position of the oligonucleotidethat results in the oligonucleotide being substantially less extendablewhen the 3′-most nucleotide of the oligonucleotide is not complementaryto the corresponding nucleotide of a target/template nucleic acid. Insome embodiments, at least one of the one or more groups is attached toa nucleotide at or near the 3′-nucleotide. In some embodiments, at leastone of the one or more groups is attached to one of the ten 3′-mostnucleotides. In other words, in embodiments of this type, at least oneof the one or more specificity enhancing groups may be attached to the3′-most nucleotide or any of the next nine contiguous nucleotides in the5′-direction. In some embodiments, at least one of the one or moregroups is attached to one of the five 3′-most nucleotides. In someembodiments, the group may be a label, preferably a label whichundergoes a detectable change in an observable property upon becomingpart of a double stranded molecule, (e.g. by hybridizing to anothernucleic acid molecule). In some embodiments, at least a portion of saidoligonucleotide is hybridized to at least a portion of said nucleic acidmolecule. In some embodiments, the oligonucleotide is capable of forminga hairpin. In some embodiments, the oligonucleotide is in the form of ahairpin. The conditions of incubation preferably include one or morepolymerase enzymes such as Tsp DNA polymerase (available from LifeTechnologies, Inc. Rockville Md.).

The present invention provides a method of synthesizing one or morenucleic acid molecules, comprising (a) contacting at least one target ortemplate nucleic acid molecule with at least one oligonucleotide of theinvention, wherein at least a portion of said oligonucleotide is capableof hybridizing with at least a portion of said target/template nucleicacid molecule (said oligonucleotide preferably comprises at least onespecificity enhancing group and/or label); and (b) incubating the targetnucleic acid and oligonucleotide mixture under conditions sufficient tocause the extension of the oligonucleotide when the 3′-most nucleotideor nucleotides of the oligonucleotide are base paired (e.g. hybridized)to said target nucleic acid molecule. In another aspect, the inventionprovides a method for reduced synthesis of one or more nucleic acidmolecules, comprising (a) contacting at least one target or templatenucleic acid molecule with at least one oligonucleotide of theinvention, wherein at least a portion of said oligonucleotide is capableof hybridizing with at least a portion of said target/template nucleicacid molecule (said oligonucleotide preferably comprises at least onespecificity enhancing group and/or label), and (b) incubating thetarget/template nucleic acid molecule and oligonucleotide mixture underconditions sufficient to prevent or inhibit extension of theoligonucleotide when the 3′-most nucleotide or nucleotides of theoligonucleotide does not base pair (e.g., does not hybridize) with thenucleotide at the specific position or positions of the target/templatenucleic acid molecule. In a preferred aspect, the results of thesynthesis of the above first method is compared to the lack or reducedlevel of synthesis in the above second method. In a preferred aspect,the conditions of the first method are conducted such that all or aportion of the target nucleic acid molecule is amplified, while theconditions in the second method are conducted such that a target nucleicacid molecule is not amplified or amplified at a reduced level and/or aslower rate compared to the amplified target nucleic acid moleculeproduced by the first method. In some embodiments, the specificityenhancing group is a fluorescent moiety. In some embodiments, the groupis attached to a nucleotide at or near the 3′-nucleotide. In someembodiments, the group is attached to one of the ten 3′-mostnucleotides. In other words, in embodiments of this type, the group maybe attached to the 3′-most nucleotide or any of the next nine contiguousnucleotides in the 5′-direction. In some embodiments, the group may be alabel, preferably a label which undergoes a detectable change in anobservable property upon becoming part of a double stranded molecule,(e.g. by hybridizing to another nucleic acid molecule). In someembodiments, at least a portion of said oligonucleotide is hybridized toat least a portion of said nucleic acid molecule. In some embodiments,the oligonucleotide is capable of forming a hairpin. In someembodiments, the oligonucleotide is in the form of a hairpin. Theincubation conditions preferably include one or more polymerase enzymessuch as Tsp DNA polymerase available from Life Technologies, Rockville,Md.

The present invention provides a method of quenching fluorescence from afluorescent moiety, comprising the step of attaching the fluorescentmoiety to an oligonucleotide, wherein the oligonucleotide is capable ofassuming a conformation in which the oligonucleotide quenches thefluorescence of the fluorescent moiety. In some embodiments, theconformation is a hairpin.

The present invention also relates to kits for the detection ormeasurement of nucleic acid molecules or for polymerase activity in asample. Such kits may also be designed to detect/quantitate nucleic acidmolecules of interest during or after nucleic acid synthesis oramplification reactions. Such kits may be diagnostic kits where thepresence of the nucleic acid is correlated with the presence or absenceof a disease or disorder. The invention also relates to kits forcarrying out extension, synthesis and/or amplification reactions of theinvention and to kits for making the compositions of the invention.

In specific embodiments, the kits comprise one or more oligonucleotidesof the invention (including primers and/or probes). The kit can furthercomprise additional components for carrying out thedetection/quantification assays or other methods of the invention. Suchkits may comprise one or more additional components selected from thegroup consisting of one or more polymerases (e.g., DNA polymerases andreverse transcriptases), one or more nucleotides, one or more bufferingsalts (including nucleic acid synthesis or amplification buffers), oneor more control nucleic acid target molecules (to act as positivecontrols to test assay or assist in quantification of the amount ofnucleic acid molecules in unknown samples), one or more quenchers(single stranded binding proteins, blocking oligonucleotides etc.),instructions for carry one out the methods of the invention and thelike. Control nucleic acid molecules are preferably provided in the kitsof the invention at known concentrations to establish control samples ofknown amounts of target molecules to assist one in establishing theamount of nucleic acid molecule of interest in an unknown sample. Thus,the measurement of activity of the labeled oligonucleotide for a knownsample may be compared to such measurement for an unknown sample toquantify the amount of the target nucleic acid molecule in the unknownsample. The kits of the invention preferably comprise a container (abox, a carton, or other packaging) having in close confinement thereinone and preferably more containers (tubes, vials and the like) whichcomprise various reagents for carrying out the methods of the invention.The reagents may be in separate containers or may be combined indifferent combinations in a single container. Such kits of the inventionmay further comprise instructions or protocols for carrying out themethods of the invention and optionally may comprise an apparatus orother equipment for detecting the detectable labels associated with theoligonucleotides of the invention.

It will be readily apparent to one of ordinary skill in the relevantarts that other suitable modifications and adaptations to the methodsand applications described herein are obvious and may be made withoutdeparting from the scope of the invention or any embodiment thereof.Having now described the present invention in detail, the same will bemore clearly understood by reference to the following examples, whichare included herewith for purposes of illustration only and are notintended to be limiting of the invention.

Example 1 Preparation of Oligonucleotides

Oligonucleotides may be prepared using any known methodology. In somepreferred embodiments, oligonucleotides may be synthesized on solidsupports using commercially available technology. Oligodeoxynucleotideswere synthesized using DNA synthesizer-8700 (Milligen/Biosearch).Fluorescent moieties may be incorporated into the oligonucleotides ofthe present invention using any conventional technology. For example,fluorescent labels may be incorporated into nucleoside phosphoramiditesand directly incorporated into the oligodeoxynucleotides duringautomated chemical synthesis. In some preferred embodiments, themodified nucleotide may be a fluorescein-dT phosphoramidite (GlenResearch Cat #10-1056) which may be inserted into designated positionduring chemical synthesis of oligonucleotide. 5′-fluoresceinphosphoramidite (FAM) (Glen Research Cat # 10-5901) and 3′-TAMRA-CPG 500(Glen Research cat #20-5910) were used to add the indicated labels tothe 5′ and 3′-end respectively of the oligodeoxynucleotide duringchemical synthesis. Alternatively, a nucleotide containing a reactivefunctional moiety may be incorporated into the oligonucleotide duringsynthesis. After the completion of the synthesis and removal of theoligonucleotide from the solid support, the reactive functional moietymay by used to couple a fluorescent moiety containing molecule to theoligonucleotide. In some preferred embodiments, the reactive functionalmoiety may be an amino-modified C6-dT (Glen Research Catalog #10-1039)which may be inserted into designated position during chemical synthesisof oligonucleotide and used for further modification. The furthermodification may include the incorporation of a fluorescently labeledmolecule. In some preferred embodiments, the fluorescently labeledmolecule may be a 6-carboxyfluorescein succinimidyl ester (6-FAM, SE,cat# C6164 Molecular Probes), Fluorescein-5-isothiocyanate (FITC)(Molecular probe cat# F-1907), 5-(6-)-carboxytetramethylrhodamine(TAMRA) succinimidyl ester (Molecular Probes), or BODIPY 530/550succinimidyl ester (Molecular Probes).

All labeled oligonucleotides may be purified using reverse-phase HPLC,for example, on a C-18 column using a gradient of acetonitrile in 0.2 Mtriethyl ammonium acetate.

Oligonucleotides of the invention may be synthesized by standard methodsknown in the art, e.g. by use of an automated DNA synthesizer (such asare commercially available from Biosearch, Applied Biosystems, etc.). Asexamples, phosphorothioate oligonucleotides may be synthesized by themethod of Stein et al. Nucl. Acids Res. 16:3209 (1988),methylphosphonate oligonucleotides can be prepared by use of controlledpore glass polymer supports (Sarin et al., Proc. Natl. Acad. Sci. USA85:7448-7451 (1988)). Oligonucleotides may also be prepared by standardphosphoramidite chemistry; or by cleavage of a larger nucleic acidfragment using non-specific nucleic acid cleaving chemicals or enzymesor site-specific restriction endonucleases. Labeled oligonucleotides ofthe invention may also be obtained commercially from Life Technologies,Inc. or other oligonucleotide manufactures.

A preferable method for synthesizing oligonucleotides is by using anautomated DNA synthesizer using methods known in the art. Once thedesired oligonucleotide is synthesized, it is cleaved from the solidsupport on which it was synthesized and treated, by methods known in theart, to remove any protecting groups present. The oligonucleotide maythen be purified by any method known in the art, including extractionand gel purification. The concentration and purity of theoligonucleotide may be determined by examining the oligonucleotide thathas been separated on an acrylamide gel, or by measuring the opticaldensity at 260 nm in a spectrophotometer.

Oligonucleotides of the invention may be labeled during chemicalsynthesis or the label may be attached after synthesis by methods knownin the art. In a specific embodiment, the label moiety is a fluorophore.Suitable moieties that can be selected as fluorophores or quenchers areset forth in Table 1.

TABLE 1 4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acidacridine and derivatives: acridine acridine isothiocyanate5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS)4-amino-N-3-vinylsulfonyl)phenylnaphthalimide-3,5 disulfonate (LuciferYellow VS) N-(4-anilino-1-naphthyl)maleimide anthranilamide BrilliantYellow coumarin and derivatives: 7-amino-4-methylcoumarin (AMC, Coumarin120) 7-amino-4-trifluoromethylcouluarin (Coumaran 151) cyanosine4′,6-diaminidino-2-phenylindole (DAPI)5′,5″-dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red)7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarindiethylenetriamine pentaacetate4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid5-dimethylaminonaphthalene-1-sulfonyl chloride (DNS, dansyl chloride)4-(4′-dimethylaminophenylazo)benzoic acid (DABCYL)4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC) eosin andderivatives: eosin eosin isothiocyanate erythrosin and derivatives:erythrosin B erythrosin isothiocyanate ethidium fluorescein andderivatives: 5-carboxyfluorescein (FAM)5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF)2′7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE) fluoresceinfluorescein isothiocyanate QFITC (XRITC) fluorescamine IR144 IR1446Malachite Green isothiocyanate 4-methylumbelliferone orthocresolphthalein nitrotyrosine pararosaniline Phenol Red B-phycoerythrino-phthaldialdehyde pyrene and derivatives: pyrene pyrene butyratesuccinimidyl 1-pyrene butyrate Reactive Red 4 (Cibacron ™ Brilliant Red3B-A) rhodamine and derivatives: 6-carboxy-X-rhodamine (ROX)6-carboxyrhodamine (R6G) lissamine rhodamine B sulfonyl chloriderhodamine (Rhod) rhodamine B rhodamine 123 rhodamine X isothiocyanatesulforhodamine B sulforhodamine 101 sulfonyl chloride derivative ofsulforhodamine 101 (Texas Red) N,N,N′,N′-tetramethyl-6-carboxyrhodamine(TAMRA) tetramethyl rhodamine tetramethyl rhodamine isothiocyanate(TRITC) riboflavin rosolic acid terbium chelate derivative

One of ordinary skill in the art can easily determine, using art-knowntechniques of spectrophotometry, which of the above identifiedfluorophores or combinations thereof can be used in accordance with theinvention. Oligonucleotides are preferably modified during synthesis,such that a modified T-base is introduced into a designated position bythe use of Amino-Modifier C6 dT (Glen Research), and a primary aminogroup is incorporated on the modified T-base, as described by Ju et al.(Proc. Natl. Acad. Sci., USA 92:4347-4351 (1995)). These modificationsmay be used for subsequent incorporation of fluorescent dyes intodesignated positions of the labeled oligonucleotides.

In yet another embodiment, the labeled oligonucleotides may be furtherlabeled with any other art-known detectable marker, includingradioactive labels such as ³²P, ³⁵S, ³H, and the like, or with enzymaticmarkers that produce detectable signals when a particular chemicalreaction is conducted, such as alkaline phosphatase or horseradishperoxidase. Such enzymatic markers are preferably heat stable, so as tosurvive the denaturing steps of the amplification or synthesis process.

Oligonucleotides may also be indirectly labeled by incorporating anucleotide linked covalently to a hapten or to a molecule such asbiotin, to which a labeled avidin molecule may be bound, or digoxygenin,to which a labeled anti-digoxygenin antibody may be bound.Oligonucleotides may be supplementally labeled during chemical synthesisor the supplemental label may be attached after synthesis by methodsknown in the art.

The sequences of the primers used in the following specific examples areprovided in Table 2.

TABLE 2 Oligo A internally   5′-cct tct cat ggt labeled with fluoresceinggc tgT aga ac (SEQ ID NO: 1) Oligo B 5′-labeled  5′-Cct tct cat ggt with fluorescein ggc tgt aga ac (SEQ ID NO: 2) Oligo C complement 5′-gtt cta cag cca  to oligo A and B cca tga gaa gg (SEQ ID NO: 3)Oligo D. 3′-labeled  5′-ggg gct gcg act  with TAMRA gtg ctc cgg cA(SEQ ID NO: 4) Oligo E. complement  5′-tgc cgg agc aca  to oligo Dgtc gca gcc cc (SEQ ID NO: 5) Oligo F. 5′-labeled  5′-Aat aat agg atg with fluorescein agg cag ga (SEQ ID NO: 6) Oligo G. 5′-labeled 5′-Aat aat agg atg  with BODIPY 530/550 agg cag ga (SEQ ID NO: 7)Oligo H complement to  5′-tcc tgc ctc atc  Oligo F and Oligo Gcta tta tt (SEQ ID NO: 8) Oligo I forward  5′-gag ttg acc gta primer for IL4 aca gac atc tt (SEQ ID NO: 9) Oligo J. forward primer 5′-ggc att gcc gac  for b-actin internally agg aTg tag aagLabeled with fluorescein (SEQ ID NO: 10) Oligo K. reverse 5′-ggg ccg gac tcg  primer for b-actin tca tac (SEQ ID NO: 11)Oligo L. forward primer   5′-ggt tgT aga gca  for b-actin labeled withctc agc aca atg aag Fluorescein through the tail- a (SEQ ID NO: 12)Oligo 1 IL 4 forward primer 5'-gag ttg acc gta  aca gac atc tt(SEQ ID NO: 13) Oligo 2 IL 4 reverse  5′-cct tct cat ggt  primer, 297WTggc tgt aga ac (SEQ ID NO: 14) Oligo 3 IL 4 reverse  5′-cct tct cat ggt primer, 297MUT ggc tgt aga at (SEQ ID NO: 15) Oligo 4 IL 4 reverse 5′-gtg tcc ttc tca  primer, 300WT tgg tgg ctg tag (SEQ ID NO: 16)Oligo 5 IL 4 reverse  5′-gtg tcc ttc tca  primer, 300MUT tgg tgg ctg tat(SEQ ID NO: 17) Oligo 6 IL 4 reverse  5′-cct tct cat ggt printer, 297WT-Fluo ggc tgT aga ac (SEQ ID NO: 18) Oligo 7 IL 4 reverse 5′-cct tct cat ggt  primer, 297MUT-Fluo ggc tgT aga at (SEQ ID NO: 19)Oligo 8 IL 4 reverse  5′-gtg tcc ttc tca  primer, 300WT-Fluotgg tgg ctg Tag (SEQ ID NO: 20) Oligo 9 IL 4 reverse 5′-gtg tcc ttc tca  primer, 300MUT-Fluo tgg tgg ctg Tat (SEQ ID NO: 21)Oligo 10 RDS reverse  5′-cta ccg ggt gtc  primer-Fluo tgt gtc tcg gTa g(SEQ ID NO: 22) Oligo 11 RDS forward  5′-cgt acc tgg cta primer, C-allele tct gtg tc (SEQ ID NO: 23) Oligo 12 RDS forward 5′-cgt acc tgg cta  primer, T-allele tct gtg tt (SEQ ID NO: 24)Oligo 13 RDS  5′-gac acc tgg cta  forward primer, tct gtg tcC-allele/hairpin (SEQ ID NO: 25) Oligo 14 RDS  5′-aac aca cct ggc forward primer, tat ctg tgt t T-allele/hairpin (SEQ ID NO: 26)Oligo 15 IL 4 reverse  5′-cta cag tcc ttc  primer/hairpintca tgg tgg ctg tag (SEQ ID NO: 27) Oligo 16 b-globin forward 5′-ctt cct gag agc  primer/linear-A cga act gta gtg a (SEQ ID NO: 28)Oligo 17 b-globin reverse  5′-aca tgt att tgc  primer/linear-Aatg gaa aac aac tc (SEQ ID NO: 29) Oligo 18 b-globin forward 5′-tca cta ctt cct  primer/hairpin-A gag agc cga act gta  gtg a(SEQ ID NO: 30) Oligo 19 b-globin reverse  5′-gag ttg tac atg primer/hairpin-A tat ttg cat gga aaa  caa ctc (SEQ ID NO: 31)Oligo 20 b-globin forward  5′-gct cag aat gat  primer/linear-Bgtt tcc acc ttc (SEQ ID NO: 32) Oligo 21 b-globin reverse 5′-aaa tca tac tag  primer/linear-B ctc acc agc aat g (SEQ ID NO: 33)Oligo 22 b-globin forward  5′-gaa ggt gct cag  primer/hairpin-Baat gat gtt tcc acc  ttc (SEQ ID NO: 34) Oligo 23 b-globin reverse 5′-cat tgc aaa tca  primer/hairpin-B tac tag ctc acc agc  aat g(SEQ ID NO: 35) Oligo 24 NF 1355 forward  5′-tgg cag ttg aat primer/linear gcc aag taa t (SEQ ID NO: 36) Oligo 25 NF 1355 reverse 5′-aca gcc act gtg  primer/linear ccc agg tc (SEQ ID NO: 37)Oligo 26 NF 1355 forward  5′-att act tgg cag  primer/hairpinttg aat gcc aag taa  t (SEQ ID NO: 38) Oligo 27 NF 1355 reverse 5′-gac ctg aca gcc  primer/hairpin act gtg ccc agg tc (SEQ ID NO: 39)Oligo 28 NF 1616 forward 5′-att tca tgg ggg  primer/linearaaa caa aga tg (SEQ ID NO: 40) Oligo 29 NF 1616 reverse 5′-ata cct gcg ctc  primer/linear acc aca gg (SEQ ID NO: 41)Oligo 30 NF 1616 forward  5′-cat ctt tat ttc  primer/hairpinatg ggg gaa aca aag  atg (SEQ ID NO: 42) Oligo 31 NF 1616 reverse 5′-cct gtg_ata cct  primer/hairpin gcg ctc acc aca gg (SEQ ID NO: 43)

The nucleotide to which the fluorescent moiety is attached is indicatedby a bold capital letter.

Example 2 PCR Targets and Conditions

Those skilled in the art will appreciate that any nucleic acid that canbe amplified by PCR may be used in the practice of the presentinvention.

Examples of suitable nucleic acids include, but are not limited to,genomic DNAs, cDNAs and cloned PCR products. The practice of the presentinvention is not limited to use with DNA molecules. For example, mRNAmolecules may be used as templates for an amplification reaction byfirst conducting a first strand synthesis reaction using techniques wellknown in the art. The present invention has been exemplified using cDNAsfor IL4 and b-actin synthesized using total mRNA from the correspondingcells and SuperScript™ System for the First Strand cDNA Synthesis (GibcoBRL, cat #18089-011) according to the manufacturer's manual. IL4 andb-actin cDNAs were amplified and cloned into pTEPA plasmid according toGibco BRL manual (cat #10156-016).

The selection of suitable PCR conditions is within the purview ofordinary skill in the art. Those skilled in the art will appreciate thatit may be necessary to adjust the concentrations of the nucleic acidtarget, primers and temperatures of the various steps in order tooptimize the PCR reaction for a given target and primer. Suchoptimization does not entail undue experimentation. In the specificexamples provided herein, PCR was performed in 25 μl of PLATINUM® TaqReaction Buffer with 0.5 un of PLATINUM® Tag, 0.2 mM dNTPs, 0.2 μMforward and reverse primers, and 1.75 mM MgCl₂ using 10⁴-10⁶ copies oftarget. PLATINUM® Tsp was used under the same conditions. Thermalcycling was performed on 9600 or ABI PRIZM™ 7700 Sequence Detector(Perkin Elmer) with 4 min denaturation at 94° C., followed by 35-40cycles: 15 sec at 94° C., 30 sec at 55° C. and 40 sec at 72° C. Intwo-step PCR cycling conditions were 15 sec at 94° C. and 30 sec at 65°C.

Example 3 Detection of Nucleic Acids

Nucleic acids may be detected by any conventional technology. In somepreferred embodiments, the nucleic acid to be detected may be a PCRproduct and may be detected either by agarose gel electrophoresis or byhomogeneous fluorescence detection method as described in U.S.provisional patent application Ser. No. 60/139,890, filed Jun. 22, 1999.In this method fluorescent signal is generated upon the incorporation ofthe specifically labeled primer into the PCR product. The method doesnot require the presence of any specific quenching moiety or detectionoligonucleotide. In some preferred embodiments, the detectionoligonucleotides are capable of forming a hairpin structure and arelabeled with fluorescein attached close to the 3′-end.

The fluorescent measurements were performed in the PCR reaction bufferusing on ABI PRIZM™7700 Sequence Detector, fluorescent plate reader(TECAN) or Kodak EDAS Digital Camera. Excitation/emission wavelengthswere 490 nm/520 nm for fluorescein and 555 nm/580 nm for TAMRA.

Example 4 Fluorescence Signal of Oligonucleotide Internally Labeled withFluorescein Increases Upon its Hybridization to the ComplementaryOligonucleotide

Two oligonucleotides of the same sequence were labeled with fluoresceineither internally on T-base (oligo A (SEQ ID NO:1)), or at the 5′-end(oligo B (SEQ ID NO:2)) as described above. 10 pmoles of eacholigonucleotide was hybridized to the complementary oligo C (SEQ IDNO:3) (50 pmoles) in 0.05 ml of the PCR buffer, heated at 70° C. for 2min and cooled to 25° C. Melting curves between 25 and 95° C. weredetermined on ABI PRIZM™ 7700 Sequence Detector.

As shown in FIG. 2, in case of internally labeled Oligo A (SEQ ID NO:1),a fluorescence signal increases as a result of presence of thenon-labeled complementary oligonucleotide. That means the signalincrease was caused by the formation of the double-stranded structure.In contrast, when the fluorescein was present on the 5′-end of the samesequence (Oligo B (SEQ ID NO:2)), fluorescence signal decreased uponhybridization.

Example 5 Oligodeoxynucleotide Labeled with TAMRA on its 3′-End,Increases the Fluorescence Signal Upon Hybridization

20 pmoles of Oligo D (SEQ ID NO:4) 3′-labeled with TAMRA as describedabove was annealed to 100 pmoles of complementary non-labeledoligodeoxynucleotide (Oligo E (SEQ ID NO:5)) in 0.5 ml of the PCRBuffer. Fluorescence emission spectrum was detected onspectrofluorimeter with 555 nm excitation.

As shown in FIG. 3, a significant increase of the signal was observedupon hybridization, indicating that the proposed method can be appliedto different fluorophores. The curve labeled buffer shows thefluorescence as a function of wavelength of the buffering solution. Thecurve labeled single-stranded shows the results obtained with thesingle-stranded version of oligo D (SEQ ID NO:4) alone. When anon-complementary oligonucleotide was added to oligo D (SEQ ID NO:4) aslight decrease in signal was observed (+non-complement). Whencomplementary oligonucleotide oligo E (SEQ ID NO:5) was added, a largeincrease in fluorescence was observed (+complement).

Example 6 Oligodeoxynucleotide 5′-Labeled with BODIPY 530/550 Increasesthe Fluorescence Signal Upon Hybridization

In examples 4 and 5 oligonucleotides internally labeled with fluoresceinand 3′ labeled with TAMRA were shown to increase the fluorescenceintensity upon hybridization to the complementary oligonucleotide. Incontrast, oligonucleotides 5′-labeled with fluorescein demonstratedfluorescence quenching upon hybridization (see example 4 and [Cardulloet al, 1988, PNAS 85, 8790-8794; Wu et al. 1998, U.S. Pat. No.5,846,729]).

However, there are some dyes that can show an enhancement of thefluorescence intensity upon hybridization even though they are locatedat the 5′ position of an oligonucleotide. For example, anoligodeoxynucleotide labeled at the 5′ end with BODIPY 530/550 shows anincrease fluorescence intensity upon hybridization.

The same oligodeoxynucleotide sequence was 5′-labeled with fluorescein(Oligo F (SEQ ID NO:6)) or BODIPY 530/550 (Oligo G (SEQ ID NO:7)). 20pmoles of each labeled oligonucleotide was annealed to 100 pmoles ofcomplementary non-labeled oligodeoxynucleotide (Oligo H (SEQ ID NO:8))in 0.5 ml of the PCR Buffer. Fluorescence emission spectrum was detectedon spectrofluorimeter with 490 nm excitation in case of fluorescein and538 nm excitation in case of BODIPY.

As shown in FIG. 4, a significant increase of the signal uponhybridization in case of BODIPY dye was observed, in contrast, adecrease in the signal was observed upon hybridization of a fluoresceincontaining oligonucleotide.

The results shown in Examples 4, 5 and 6 demonstrate that thefluorescent properties of a given fluorophore, in particular thefluorescent intensity, can be affected upon hybridization withoutsignificant shift of the emission spectrum as a result of the point ofattachment of the fluorphore to a given oligonucleotide, i.e., internal,3′ and 5′.

Example 7 Quantitative PCR of IL4 cDNA Using Primer Internally Labeledwith Fluorescein

Fluorescein-dT was directly incorporated into the sequence of IL-4primer during chemical synthesis using the methods described above. Theresulting oligonucleotide (Oligo A (SEQ ID NO:1)) was used as a reverseprimer for IL4 cDNA amplification. Quantitative PCR using reverse primer(Oligo A (SEQ ID NO:1)) and forward primer (Oligo I (SEQ ID NO:9)) wasperformed as described above in the presence of varying amounts of thetemplate DNA. 10⁷, 10⁶, 10⁵, 10⁴, 10³, 10², 10 and 0 copies of thecloned IL4 a target were used per reaction along with four samples ofunknown concentration of the target. As shown in FIG. 5, all dilutionsof the DNA target can be detected with extremely high accuracy.

The results of this experiment demonstrate that although no quencher ispresent in the structure of labeled oligonucleotide, it can besuccessfully used in quantitative PCR.

Example 8 Real-Time PCR of IL4 cDNA Using Primer Post-SyntheticallyLabeled with FITC

Reverse primer for IL4 (Oligo A (SEQ ID NO:1)) was synthesized andlabeled post-synthetically as described above. Amplification wasperformed with 10⁶, 10⁴, 10² and 0 copies of nucleic acid target asdescribed in the previous example. As shown in FIG. 6, all dilutions ofthe DNA target can be detected.

The experimental results in preceding examples demonstrate thatdifferent methods of the labeling of oligonucleotides can be used forachieving the same result. Also, since two methods of synthesis providedifferent structures of the linker arm between oligonucleotide andfluorophore, different linker arms can be used to attach fluorophore inthe proposed method.

Example 9 Real-Time PCR of b-Actin cDNA with a Primer Internally Labeledwith Fluorescein

Fluorescein-dT was directly incorporated into the sequence of theforward primer for human b-actin cDNA (Oligo J (SEQ ID NO:10)) duringchemical synthesis. This oligonucleotide and unlabeled reverse primer(Oligo K (SEQ ID NO:11)) were used for the amplification of b-actincDNA. cDNA target was obtained by reverse transcription of HeLa cellmRNA and also a cloned cDNA fragment (10⁷, 10⁵ and 0 copies perreaction). Quantitative PCR was performed as described above. As shownin FIG. 7, all dilutions of the DNA target can be detected.

The results of this experiment demonstrate that different targets can bedetected using the proposed method.

Example 10 Real-Time PCR of b-Actin cDNA with a Primer InternallyLabeled Through a “Tag” Sequence Non-Complementary to the Target

All the above experiments showed that the label could be incorporatedinto the sequence of oligonucleotide complementary to the target nucleicacid. However, the same result can be obtained if the label is presenton a non-complementary tag sequence attached to the 5′-end of a PCRprimer. In this case a signal will be generated after this tailed primeris copied and incorporated into the double-stranded PCR product. Thisapproach was demonstrated in the b-actin PCR.

Oligodeoxynucleotide (Oligo L (SEQ ID NO:12)) was synthesized withFluorescein-dT directly incorporated into the structure of 9-nucleotidetail, non-complementary to the target. This tail was added to the 5′-endof the b-actin forward primer. Oligo L (SEQ ID NO:12) and unlabeledreverse primer (Oligo K (SEQ ID NO:11)) were used to amplify b-actincDNA and 10⁶, 10⁴, and 0 copies of cloned target. As shown in FIG. 8,both cloned target and cDNA in total cDNA population can be detected.

Example 11 Allele Specific PCR with Modified Oligonucleotide Primers

The principle of allele specific PCR is presented in FIG. 9. The methodoperates on the basis of the specific amplification of a target alleleby the PCR with primers designed such that their 3′ ends are placed atthe mutation site (i. e., the 3′-most nucleotide of the primercorresponds to the mutated nucleotide in the target/template nucleicacid). When this base is complementary to that of the correspondingnucleotide of the specific allele, the target is amplified; when it isnot complementary PCR will proceed with a significant delay. The longerthe delay, the more efficiently the system can discriminate betweenalleles. In some preferred embodiments, the present invention providesoligonucleotides useful for allele specific PCR which oligonucleotidescomprise a specificity enhancing group that improves discriminationbetween alleles.

Allele specific PCR was performed using regular PCR primers and theprimers labeled with fluorescein at a base close to the 3′-end. Twopositions of the IL4 cDNA were chosen for detection, C297 and G300. Foreach position two PCRs were performed using the same forward primer(Oligo 1 (SEQ ID NO:13)) and different reverse primers: wild type (WT),complementary to the target, or mutant (MUT) with a mismatch at the3′-end. The sequences of the primers used are provided in Table 2. Eachof these allele specific primers was synthesized with and withoutchemical modification on a T-base close to the 3′-end. The primers usedwere 297 WT-primer complementary to the C-allele at position 297 (Oligo2 (SEQ ID NO:14)), 297 MUT- same primer with C-T mutation at the 3′-end(Oligo 3 (SEQ ID NO:15)), 300 WT- primer complementary to the C-alleleat position 300 (Oligo 4 (SEQ ID NO:16)) and 300 MUT- same primer withG-T mutation at the 3′-end (Oligo 5 (SEQ ID NO:17)). Oligonucleotides 6,7, 8, 9 (SEQ ID NOs:18, 19, 20, 21, respectively) correspond tooligonucleotides 2, 3, 4, 5 (SEQ ID NOs:14, 15, 16, 17, respectively)with fluorescein attached to the designated T-base.

Three step PCR was performed for 40 cycles with Platinum Taq™ asdescribed above and the results are shown in FIG. 10. Reverse primerswith their 3′-end at positions 297 or 300 were either complementary tothe target (WT) or had a 3′ mutation (MUT). Lanes 1 through 4 show theresults obtained with primers modified with fluorescein as a specificityenhancing group; lanes 5 through 8 show the results obtained withunmodified primers. Lanes 1 and 5 show the results using the primer 297WT; lanes 2 and 6 show the results using the primer 297 MUT; lanes 3 and7 show the results using primer 300 WT; lanes 4 and 8 show the resultsusing primer 300 MUT. A comparison of lanes 2 and 6 and a comparison oflanes 4 and 8 show that the presence of a modification allowsdiscrimination that is almost complete after 40 cycles. The practice ofthe present invention is not limited to the use of fluorescein, similarresults were obtained with TAMRA as a specificity enhancing group (datanot shown).

Example 12 Allele Specific PCR with Hairpin Oligonucleotide Primers

In some preferred embodiments, the primers of the present invention maybe modified such that they assume a hairpin structure. This may beaccomplished by adding one or more bases to the 5′-terminal of theoligonucleotide which bases are selected to be complementary to thebases at the 3′-terminal of the oligonucleotide. In some preferredembodiments, at least one to about 20 contiguous nucleotides are addedto the 5′ end of the oligonucleotide that are complementary to the atleast one to 20 contiguous nucleotides present in the 3′-end of theoligonucleotide. In a preferred embodiment, from one to about 10nucleotides are added to the 5′-end of the oligonucleotide, thenucleotides selected such that they are complementary to the at leastone to about 10 contiguous nucleotides present in the 3′-end of theoligonucleotide. In another preferred embodiment, from one to about 5nucleotides are added to the 5′-end of the oligonucleotide, thenucleotides selected such that they are complementary to the at leastone to about 5 contiguous nucleotides present in the 3′-end of theoligonucleotide.

The present invention is based upon the surprising result that themutation discrimination can be improved through the secondary structureof the allele specific primers. This feature is exemplified usingprimers specific for the RDS gene. Forward primers for the RDS gene hadtheir 3′ ends located at position 558, the site of a C/T polymorphism.The DNA target contained the C-allele. The reverse primer was the samefor both alleles and contained the label that permitted homogeneousdetection of amplification in real time (Oligo 10 (SEQ ID NO:22)).Forward allele specific primers were either of the conventional linearstructure (Oligo 11, 12 (SEQ ID NOs:23, 24, respectively)) or had thehairpin structure (Oligo 13, 14 (SEQ ID NOs:25, 26, respectively)).Hairpin primers consisted of the target-specific sequence and a shorttail complementary to the 3′-fragment of the primer. Three step PCR wasperformed with Platinum Taq™ DNA polymerase on PRIZM 7700 as describedabove. The results in FIG. 11 show that the blunt-end hairpin structureof the primer significantly improves mutation discrimination. Theprimers of the invention were used to discriminate between the C and theT allele of human RDS gene by allele-specific PCR with Platinum Taq™ DNApolymerase using the same fluorescent reverse primer (Oligo 10 (SEQ IDNO:22)) and different allele specific forward primers. The primers usedwere designated L-C for the linear primer specific for C-allele (Oligo11 (SEQ ID NO:23)), L-T for the linear primer specific for T-allele(Oligo 12 (SEQ ID NO:24)), H-C for the hairpin primer specific forC-allele (Oligo 13 (SEQ ID NO:25)) and H-T for the hairpin primerspecific for T-allele (Oligo 14 (SEQ ID NO:26)). A comparison of thereal time fluorescence of the reactions is plotted as a function of thecycle number. The linear T mismatched primer generated a signal that wasdetectable well before the hairpin T mismatched primer signal. Thisdemonstrates that the discrimination between the alleles was improved byincorporating the 3′-terminal of the primer into a hairpin

Another example of allele specific PCR using hairpin primers is shown inFIG. 12. Here two genomic DNA samples were tested by two step PCR. Oneof the samples was known to have a 558C-allele of RDS gene, another the558T allele. All forward primers were hairpin primers and fluorescentreverse primer was used for the detection. Curve 1 was obtained with theC-primer with C-target DNA; curve 2 was obtained using the C-primer withT-target DNA; curve 3 was obtained using C-primer with no target DNA(negative control); curve 4 was obtained using the T-primer withT-target DNA; curve 5 was obtained using T-primer with C-target DNA;curve 6 was obtained using T-primer with no target (negative control).

The results demonstrate that only C-allele with C-specific primers andT-allele with 1-specific primers gave a positive signal when hairpinprimers were used. No increase of fluorescence was detected when theprimer had a 3′-mismatch. No signal was generated in the absence oftarget. As shown in FIG. 13, the alleles can be detected with the samehigh level of specificity not only in real time but also at the endpoint. Fluorescent reverse primer was used for the detection. 1, 3, 5C-specific primers, 2, 4, 6 T-specific primers, 1 and 2 C allele targetDNA, 3 and 4 T allele target DNA, 5 and 6 no DNA (negative controls).Panel A shows a bar graph of the fluorescence obtained while Panel Bshows a photograph of the reaction mixture after the amplificationreactions. End point detection is permitted by high signal/noise ratioof the detection system and can be performed using fluorescent platereader or UV transilluminator and digital camera.

Another surprising result of the use of the primers of the presentinvention is the elimination of primer dimers from the PCR reaction. Asshown in FIG. 14, the use of a hairpin oligonucleotide in the PCRreaction eliminates the formation of primer dimers. IL4 cDNA was used asa PCR target. Oligo 1 (SEQ ID NO:13) was used as a forward primer, oligo2 (SEQ ID NO:14) as a linear reverse primer and Oligo 15 (SEQ ID NO:27)as a hairpin reverse primer. PCR was performed with platinum Taq™ for 50cycles. Lanes 1, 5 contained 10⁶ copies of target; lanes 2, 6 contained10⁴ copies of target; lanes 3, 7 contained 10² copies of target; andlanes 4, 8 contained no target. Comparison of the lanes 4 and 8 showsthat primer-dimer was formed with linear reverse primer but not with thehairpin.

Example 13 Use of Mismatch Discriminating Polymerases in Allele SpecificPCR

The ability to discriminate between alleles by allele specific PCR maybe improved by using DNA polymerases modified to be substantially unableto extend an oligonucleotide when the 3′-most nucleotide of theoligonucleotide is not base paired with the target nucleic acidsequence. The preparation of such modified DNA polymerases is disclosedin WO 99/10366 and WO 98/35060. These publications disclose the cloningand mutagenesis of thermostable polymerases, in particular, thethermostable DNA polymerase isolated from Thermatoga spp. In somepreferred embodiments of the present invention, allele specific PCR isperformed using a mutant DNA polymerase derived from the DNA polymeraseof Thermotoga neopolitana (Tne). Suitable mutations include deletion ofone or more amino acids, frame shift mutations, point mutations thatresult in one or more amino acid substitutions at one or more sites inthe enzyme, insertion mutations and combinations thereof. In a preferredembodiment, the mutations may include a deletion of the first 283 aminoacids of the wild type enzyme leaving a fragment that begins withmethionine 284 (Δ283), a point mutation changing amino acid 323 fromaspartic acid to alanine (D323A) and a point mutation changing aminoacid 722 from arginine to lysine (R722K). In some preferred embodiments,the mutant Tne DNA polymerase will have at least all three mutations,i.e. will be Δ283, D323A and R722K.

Platinum Tsp™ DNA polymerase is a proprietary enzyme of LifeTechnologiesthat can be activated by temperature thus providing a hot start for PCR(U.S. Pat. Nos. 5,338,671 and 5,587,287). Here we describe a newproperty of this enzyme, increased specificity towards the base-paired3′-end of the primer. PCR was performed for 45 cycles with Platinum Tsp™or Platinum Taq™ DNA polymerase using IL4 cDNA as a target. Twopositions of the IL4 cDNA were chosen for detection, C297 and G300. Foreach position two PCR reactions were performed using the same forwardprimer (Oligo 1 (SEQ ID NO:13)) and different reverse primers. Primersequences are described in Table 1 (Oligos 1-5 (SEQ ID NOs:13-17)). Theoligonucleotides are designated wild type (WT), when the 3′-nucleotideis complementary to the target, or mutant (MUT) with a mismatch at the3′-end. The oligonucleotides used were the 297 WT primer which iscomplementary to the C-allele at position 297 (Oligo 2 (SEQ ID NO:14),lane 1), the 297 MUT primer which has the same sequence as the 297 WTprimer except for a C-T mutation at the 3′-end (Oligo 3 (SEQ ID NO:15),lane 3), the 300 WT primer which is complementary to the C-allele atposition 300 (Oligo 4 (SEQ ID NO:16), lane 2) and the 300 MUT primerwhich has the same sequence as the 300 WT primer except for a G-Tmutation at the 3′-end (Oligo 5 (SEQ ID NO:17), lane 4). As seen in FIG.15, a comparison of the results obtained with Tsp™ DNA polymerase tothose obtained with Taq™ DNA polymerase show that Platinum Tsp™ hasbetter discriminatory properties than platinum Taq™.

Example 14 Use of Hairpin Primers to Enhance Specificity of PCR

It has been unexpectedly found that the hairpin primers of the presentinvention may be used to enhance the specificity of PCR reactions.Without wishing to be bound by theory, it is believed that the abilityof the primers to form hairpin structures at temperatures around theannealing temperature of the PCR reaction makes the primers less capableof mis-priming to the target nucleic acid molecule. This increase inspecificity is not dependent upon the particular target nucleic acidtemplate and has been observed with a variety of templates. The increasein specificity will be particularly important for the amplification oftemplates that are difficult to amplify and that produce low amounts ornone of the desired amplification product in PCR reactions.

In addition to hairpin structures, any structure that sequesters the3′-end of the oligonucleotide primer may be used to practice the presentinvention. For example, the 5′-portion of the primers of the presentinvention may be provided with sequence that is capable of forming aduplex such that the 3′-end interacts with the duplex to form a triplex.In general, any primer sequence that reversibly involves the 3′-portionof the primer in a stable structure that is not capable annealing to thetemplate DNA while in that structure may be used to practice the presentinvention. In some embodiments, an oligonucleotide complementary to theprimer may be provided so as to sequester the 3′-end of the primer.Complementary oligonucleotides may be provided with a 5′-overhangingregion which may be designed to include self complementary regionscapable of forming hairpins. It is not necessary that the entire3′-portion of the primer be sequestered, so long as the portion notsequestered is not capable of mis-priming the nucleic acid template, itis sufficient to practice the present invention.

In the first experiment, a 3.6 kb fragment of the human beta-globin wasamplified from human genomic DNA using Platinum Pfx thermostablepolymerase in Pfx buffer (LifeTechnologies). Two different sets ofprimers were used. Each set of primers consisted of two primer pairs,one pair of linear primers and another pair of primers having a hairpinversion of the same gene specific primer sequence. The hairpin versionof each pair of oligonucleotides was constructed by adding bases to the5′-end of the primer sequence that are complementary to the 3′-end ofthe oligonucleotide. Typically, the number of bases added to the 5′-endis selected such that the oligonucleotide forms a hairpin attemperatures below the annealing temperature and assumes a linear format or near the annealing temperature. Those skilled in the art canreadily determine the number of nucleotides to be added to the 5′-end ofthe primer so as to control the temperature at which the primer assumesa linear form. It is not necessary that the oligonucleotides of theinvention be entirely converted to linear form at the annealingtemperature; those skilled in the art will appreciate that theoligonucleotides of the present invention may be capable of reversiblymelting and self reannealing (i.e., breathing). So long as the sequencesof the oligonucleotides of the invention are selected such that asufficient number of oligonucleotides are available to prime theextension/amplification at the annealing temperature, the sequence issuitable for use in the present invention whether or not some of theoligonucleotides remain in a hairpin form at the annealing temperature.The number of nucleotides that may be added may be from about 3nucleotides to about 25 nucleotides, or from about 3 nucleotides toabout 20 nucleotides, or from about 3 nucleotides to about 15nucleotides, or from about 3 nucleotides to about 10 nucleotides, orfrom about 3 nucleotides to about 7 nucleotides. In some preferredembodiments, from about 5 to about 8 nucleotides may be added to the5′-end of the primer oligonucleotide in order to form the hairpinoligonucleotides of the present invention. For the amplification of thebeta globin gene, two sets of primers were used. Set A oligos 16 (SEQ IDNO:28) and 17 (SEQ ID NO:29) (linear) or 18 (SEQ ID NO:30) and 19 (SEQID NO:31) (hairpin) and Set B-oligos 20 (SEQ ID NO:32) and 21 (SEQ IDNO:33) (linear) or 22 (SEQ ID NO:34) and 23 (SEQ ID NO:35) (hairpin).PCR was performed as follows: 2 minutes at 94° C. followed by 35 cyclesof: 15 seconds at 94° C. then 30 seconds at 60° C. followed by 4 minutesat 68° C. using varying amounts of template DNA. The results are shownin FIG. 16. The lanes labeled M contain molecular weight markers. Lanes1 and 2 show the results obtained using 50 ng of template DNA, lanes 3and 4 show the results obtained using 20 ng of template and lanes 5 and6 show the no DNA controls. It is clear that both linear sets of primersgenerated various mis-priming products and primer-dimers, whileamplification with the corresponding hairpin primers produced theexpected size amplification product with very little incorrect product.

Similar results were obtained during the amplification of another humangene Necrosis Factor 2(NF2). 1.3 and 1.6 kb fragments were amplifiedusing Platinum Taq DNA polymerase in PCR SuperMix (LifeTechnologies).For the amplification of the 1.3 kb fragment oligos 24 (SEQ ID NO:36)and 25 (SEQ ID NO:37) (linear) or 26 (SEQ ID NO:38) and 27 (SEQ IDNO:39) (hairpin) were used as primers. For the amplification of the 1.6kb fragment oligos 28 (SEQ ID NO:40) and 29 (SEQ ID NO:41) (linear) or30 (SEQ ID NO:42) and 31 (SEQ ID NO:43) (hairpin) were used as primers.PCR was performed on 50 ng of human genomic DNA as follows: 2 minutes at94° C. followed by 35 cycles of: 30 seconds at 94° C., 30 seconds at 62°C. and 4 minutes at 68° C. The results are shown in FIG. 17. Lane Mcontains molecular weight markers. + indicates the presence of templateDNA and − indicates the no DNA control. Lane 1 shows the results usinglinear primers for the 1.3 kb fragment in the presence of template DNA.Lane 2 shows the no DNA control for lane 1. Lane 3 shows the resultsobtained using the hairpin primer for the 1.3 kb fragment while lane 4is the no DNA control for lane 3. Lane 5 shows the results obtainedusing the linear primers for the 1.6 kb fragment while lane 6 is the noDNA control for lane 5. Lane 7 shows the results obtained using thehairpin primers for the 1.6 kb fragment while lane 8 is the no DNAcontrol for lane 7. In both instances the hairpin primers gave more andcleaner amplification products of the appropriate size than linearprimers of the same gene specific sequence.

Having now fully described the present invention in some detail by wayof illustration and example for purposes of clarity of understanding, itwill be obvious to one of ordinary skill in the art that the same can beperformed by modifying or changing the invention within a wide andequivalent range of conditions, formulations and other parameterswithout affecting the scope of the invention or any specific embodimentthereof, and that such modifications or changes are intended to beencompassed within the scope of the appended claims.

All publications, patents and patent applications mentioned in thisspecification are indicative of the level of skill of those skilled inthe art to which this invention pertains, and are herein incorporated byreference to the same extent as if each individual publication, patentor patent application was specifically and individually indicated to beincorporated by reference.

1. A composition for quantifying or detecting one or more target nucleicacid molecules in a sample comprising one or more detectably labeledoligonucleotides and one or more target nucleic acid molecules to bedetected or quantified, wherein said oligonucleotides comprise one ormore detectable labels located internally and/or at or near the 3′and/or 5′ termini of said oligonucleotides and wherein said labelundergoes a detectable change in an observable property upon becomingpart of a double stranded molecule.
 2. The composition of claim 1,wherein said detectable change is an increase or enhancement in thelevel of activity of the detectable label compared to the level ofactivity of the detectable label in the absence of said target nucleicacid molecules.
 3. The composition of claim 2, wherein said detectablelabels are selected from the group consisting of fluorescent labels,chemiluminescent labels and bioluminescent labels.
 4. The composition ofclaim 3, wherein the fluorescent label is selected from the groupconsisting of FAM, TAMRA, JOE, Rhodamine, BODIPY, R6G, ROX, and EDANS.5. The composition of claim 1, wherein said one or more detectablelabels are the same or different.
 6. The composition of claim 1, whereinone or more of said oligonucleotides comprise one or more hairpinstructures.
 7. The composition of claim 1, wherein one or more of saidoligonucleotides is hybridized to one or more of said nucleic acidmolecules.
 8. The composition of claim 1, further comprising at leastone component selected from the group consisting of one or morenucleotides, one or more DNA polymerases and one or more reversetranscriptases.
 9. The composition of claim 1, wherein said nucleic acidmolecules are RNA and/or DNA molecules.
 10. A method for thequantification or detection of one or more target nucleic acid moleculesin a sample comprising hybridizing one or more detectably labeledoligonucleotides of claim 1 with one or more molecules to be detected orquantified, and detecting the presence or absence and/or quantifying theamount of said target nucleic acid molecules.
 11. A method for thequantitation or detection of one or more nucleic acid molecules in asample during nucleic acid synthesis comprising: mixing one or morenucleic acid templates with one or more oligonucleotides of claim 1;incubating said mixture under conditions sufficient to synthesize one ormore nucleic acid molecules complementary to all or a portion of saidtemplates, said synthesized nucleic acid molecule comprising saidoligonucleotides; and detecting the presence or absence or quantifyingthe amount of said synthesized nucleic acid molecules by measuring saiddetectable label.
 12. A method for quantitation or detection of one ormore nucleic acid molecules in a sample during nucleic acidamplification comprising: mixing one or more nucleic acid templates withone or more oligonucleotides of claim 1 under conditions sufficient toamplify one or more nucleic acid molecules complementary to all or aportion of said templates, said amplified nucleic acid moleculecomprising said oligonucleotides; and detecting the presence or absenceor quantifying the amount of said nucleic acid molecules by measuringthe detectable labels of said oligonucleotides.
 13. The method of claim12, wherein said label is selected from the group consisting offluorescent labels, chemiluminescent labels and bioluminescent labels.14. The method of claim 11 or 12, wherein said detection step comprisesdetecting or measuring the level of activity of the detectable labelduring said synthesis or amplification compared to the level of activityof the detectable label in the absence of said synthesis oramplification.
 15. The method of claim 12, wherein said amplification isaccomplished by at least one method selected from the group consistingof PCR, 5-RACE, RT PCR, Allele-specific PCR, Anchor PCR, “one-sidedPCR,” LCR, NASBA, and SDA.
 16. The method of claim 13, wherein saidoligonucleotides comprise one or more fluorescent labels.
 17. The methodof any one of claim 10, 11 or 12, wherein said one or moreoligonucleotides comprise one or more hairpin structures.
 18. A methodfor amplifying a double stranded nucleic acid molecule, comprising:providing a first and second primer, wherein said first primer iscomplementary to a sequence within or at or near the 3′-termini of thefirst strand of said nucleic molecule and said second primer iscomplementary to a sequence within or at or near the 3′-termini of thesecond strand of said nucleic acid molecule; hybridizing said firstprimer to said first strand and said second primer to said second strandin the presence of one or more of the polymerases, under conditions suchthat a third nucleic acid molecule complementary to all or a portion ofsaid first strand and a fourth nucleic acid molecule complementary toall or a portion said second strand are synthesized; denaturing saidfirst and third strand, and said second and fourth strands; andrepeating the above steps one or more times, wherein one or more of theprimers comprise a detectable label internally and/or at or near its 3′and/or 5′ termini and/or comprises one or more hairpin structures. 19.The method of claim 18, wherein at least one of said primers comprisesat least one hairpin structure.
 20. A method for the quantification ordetection of nucleic acids molecules comprising: mixing one or morelabeled oligonucleotides with one or more nucleic acid molecules to bedetected or quantitated; and detecting or measuring an increase influorescence associated with said oligonucleotide hybridizing to saidnucleic acid molecules.
 21. The method of claim 20, wherein thefluorescent label is FAM.
 22. The method of claim 20, wherein thefluorescent label is TAMRA.
 23. A composition comprising one or morenucleic acid molecules and at least one oligonucleotide, wherein atleast a portion of said oligonucleotide is capable of hybridizing withat least a portion of said nucleic acid molecule and wherein saidoligonucleotide comprises a specificity enhancing group.
 24. Thecomposition according to claim 23, wherein the group is a fluorescentmoiety.
 25. The composition according to claim 23, wherein the moiety isattached to a nucleotide at or near the 3′-most terminal nucleotide. 26.The composition according to claim 23, wherein the moiety is attached toone of the ten 3′-most terminal nucleotides.
 27. The compositionaccording to claim 23, wherein the moiety is detectable.
 28. Thecomposition according to claim 23, wherein at least a portion of saidoligonucleotide is hybridized to at least a portion of said nucleic acidmolecule.
 29. The composition according to claim 23, wherein theoligonucleotide is capable of forming a hairpin.
 30. The compositionaccording to claim 23, wherein the oligonucleotide is in the form of ahairpin.
 31. A method of making a composition, comprising the steps ofproviding at least one oligonucleotide; and contacting saidoligonucleotide with at least one nucleic acid molecule, wherein atleast a portion of said oligonucleotide is capable of hybridizing withat least a portion of said nucleic acid molecule and wherein saidoligonucleotide comprises a specificity enhancing group.
 32. The methodaccording to claim 31, wherein oligonucleotide is in the form of ahairpin.
 33. A method of determining the presence of one or moreparticular nucleotides at a specific position or positions in a targetnucleic acid molecule, comprising: contacting at least one targetnucleic acid molecule having one or more nucleotides of interest at aspecific position or positions on a target nucleic acid molecule with atleast one oligonucleotide, wherein at least a portion of theoligonucleotide is capable of forming base pairs or hybridizing with atleast a portion of the target nucleic acid molecule and wherein theoligonucleotide comprises at least one specificity enhancing groupand/or one or more hairpin structures; and incubating theoligonucleotide and the target nucleic acid molecule under conditionssufficient to cause extension of the oligonucleotide when the 3′-mostnucleotide or nucleotides of the oligonucleotide base pair with thenucleotide or nucleotides at the specific position or positions of thetarget nucleic acid molecule, wherein the production of an extensionproduct indicates the presence of the particular nucleotide at thespecific position.
 34. The method according to claim 33, wherein thegroup is attached to a nucleotide near the 3′-terminal nucleotide.
 35. Amethod of determining the absence of one or more particular nucleotidesat a specific position or positions in a target nucleic acid molecule,comprising: contacting at least one target nucleic acid molecule havingone or more nucleotides of interest at a specific position or positionson the target nucleic acid molecule with at least one oligonucleotide,wherein at least one portion of the oligonucleotide is capable offorming base pairs or hybridizing with at least a portion of the targetnucleic acid molecule and wherein the oligonucleotide comprises at leastone specificity enhancing group and/or one or more hairpin structures;and incubating the oligonucleotide and target nucleic acid moleculeunder conditions sufficient to inhibit or prevent extension of theoligonucleotide when the 3′-most nucleotide or nucleotides of theoligonucleotide does not substantially base pair with the nucleotide ornucleotides of the specific position or positions of the target nucleicacid molecule, wherein the lack of or reduced production of an extensionproduct indicates the absence of the particular nucleotide at thespecific position.
 36. A method of determining the presence or absenceof one or more particular nucleotides at a specific position orpositions in a target nucleic acid molecule, comprising: contacting atleast first oligonucleotide with at least one target nucleic acidmolecule under conditions sufficient to cause extension of the firstoligonucleotide when the 3′-most nucleotide or nucleotides of theoligonucleotide base pairs with the nucleotide or nucleotides at thespecific position or positions of the target nucleic acid molecule,wherein said first oligonucleotide comprises at least one specificityenhancing group and/or at least one hairpin structure; contacting atleast a second oligonucleotide with at least one target nucleic acidmolecule under conditions sufficient to inhibit or prevent extension ofthe oligonucleotide when the 3′-most nucleotide or nucleotides of theoligonucleotide do not substantially base pair with the nucleotide ornucleotides at the specific position or positions of the target nucleicacid molecule, wherein said second oligonucleotide comprises at leastone specificity enhancing group and/or at least one hairpin structure;and comparing the level of extension or the amount of extension productaccomplished with the first oligonucleotide compared to the secondoligonucleotide.
 37. The method of claim 33, wherein said conditions aresufficient to cause amplification of all or a portion of the targetnucleic acid molecule.
 38. The method of claim 35, wherein saidconditions are sufficient to inhibit or prevent amplification of all ora portion of said target nucleic acid molecule.
 39. The method of claim33, wherein said conditions are accomplished in the presence of Tsp DNApolymerase.
 40. The method of claim 35, wherein said conditions areaccomplished in the presence of Tsp DNA polymerase.
 41. The compositionof claim 1, wherein said composition further comprises a quenchingmolecule.
 42. The composition of claim 41, wherein said quenchingmolecule is selected from the group consisting of a single strandedbinding protein and an oligonucleotide comprising at least one quenchingmoiety.
 43. The composition of claim 42, wherein said oligonucleotidecomprising at least one quenching moiety is capable of hybridizing to orbase pairing with said detectably labeled oligonucleotides.
 44. Thecomposition of claim 1, wherein said detectably labeled oligonucleotidefurther comprises one or more quenching moieties.
 45. The composition ofclaim 1, wherein said detectably labeled oligonucleotide comprises oneor more hairpin structures and further comprises one or more quenchingmoieties.
 46. The composition of claim 1, wherein at least one of saiddetectable labels and at least one of said quenching moieties is locatedwithin the stem of said hairpin structures.
 47. A method for detecting atarget nucleic acid sequence, comprising: contacting a sample containinga mixture of nucleic acid molecules with at least one oligonucleotide,the oligonucleotide capable of hybridizing with a target nucleic acidmolecule and comprises a detectable moiety, wherein the detectablemoiety undergoes a change in one or more observable property uponhybridization to the target nucleic acid molecule; and observing theobservable property, wherein a change in the observable propertyindicates the presence of the target nucleic acid sequence.
 48. A methodof determining the presence or absence of at least one particularnucleotide of interest at a specific position in a target nucleic acidmolecule, comprising: providing at least one target nucleic acidmolecule having said nucleotide of interest at a specific position;contacting said target nucleic acid molecule with at least oneoligonucleotide, wherein at least a portion of the oligonucleotide iscapable of forming base pairs or hybridizing with at least a portion ofthe nucleic acid molecule and wherein the oligonucleotide comprises atleast one specificity enhancing group and/or at least one label; andcontacting the oligonucleotide and the target nucleic acid molecule witha polymerase less able to extend the oligonucleotide when the 3′-mostnucleotide of the oligonucleotide does not base pair with the targetnucleic acid and more able to extend the oligonucleotide when the3′-most nucleotide of the oligonucleotide base pairs with the targetnucleic acid molecule.
 49. The method of claim 48, wherein thepolymerase enzyme is Tsp DNA polymerase.
 50. The method of claim 48,wherein the group is a fluorescent moiety.
 51. The method according toclaim 48, wherein the group is attached to a nucleotide at or near the3′-nucleotide.
 52. The method according to claim 48, wherein the groupis attached to one of the ten 3′-most nucleotides.
 53. The methodaccording to claim 48, wherein the group is detectable.
 54. The methodaccording to claim 48, wherein the oligonucleotide is in the form of ahairpin.
 55. A method for synthesizing or amplifying one or more nucleicacid molecules comprising: mixing one or more nucleic acid templates ortargets with one or more oligonucleotides, wherein said one or more ofsaid oligonucleotides comprises at least one hairpin structure; andincubating said mixture under conditions sufficient to synthesize oramplify one or more nucleic acid molecules complementary to all or aportion of said templates or targets.